Mutations in NOD2 are associated with fibrostenosing disease in patients with Crohn&#39;s disease

ABSTRACT

The present invention provides a method of diagnosing or predicting susceptibility to a clinical subtype of Crohn&#39;s disease characterized by fibrostenosing disease by determining the presence or absence in an individual of a fibrostenosis-predisposing allele linked to a NOD2/CARD15 locus, where the presence of the fibrostenosis-predisposing allele is diagnostic of or predictive of susceptibility to the clinical subtype of Crohn&#39;s disease characterized by fibrostenosing disease. In a method of the invention, the clinical subtype of Crohn&#39;s disease can be, for example, characterized by fibrostenosing disease independent of small bowel involvement. The invention also provides a method of optimizing therapy in an individual by determining the presence or absence in the individual of a fibrostenosis-predisposing allele linked to a NOD2/CARD15 locus, diagnosing individuals in which the fibrostenosis-predisposing allele is present as having a fibrostenosing subtype of Crohn&#39;s disease, and treating the individual having a fibrostenosing subtype of Crohn&#39;s disease based on the diagnosis.

RELATED APPLICATIONS

[0001] This application claims the benefit of priority of provisionalapplication serial No. 60/407,391, filed Aug. 30, 2002, and which isincorporated herein by reference.

ACKNOWLEDGMENT

[0002] This work was supported by grant DK46763 and DK54967 awarded byNIDDK. The United States government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The invention relates generally to the fields of genetics andautoimmune disease and, more specifically, to mutations linked to theNOD2/CARD15 gene and genetic methods for diagnosing clinical subtypes ofCrohn's disease.

[0005] 2. BACKGROUND INFORMATION

[0006] Inflammatory bowel disease (IBD) is the collective term used todescribe two gastrointestinal disorders of unknown etiology: Crohn'sdisease (CD) and ulcerative colitis (UC). The course and prognosis ofIBD, which occurs world-wide and is reported to afflict as many as twomillion people, varies widely. Onset of IBD is predominantly in youngadulthood with diarrhea, abdominal pain, and fever the three most commonpresenting symptoms. The diarrhea may range from mild to severe, andanemia and weight loss are additional common signs of IBD. Of allpatients with IBD, ten percent to fifteen percent will require surgeryover a ten year period. In addition, patients with IBD are at increasedrisk for the development of intestinal cancer. Reports of an increasingoccurrence of psychological problems, including anxiety and depression,are perhaps not surprising symptoms of what is often a debilitatingdisease that strikes people in the prime of life.

[0007] Crohn's disease is a classification representing a number ofheterogeneous disease subtypes that affect the gastrointestinal tractand produce similar symptoms. Both environmental and genetic factorslikely contribute to the etiology of such disease subtypes. Patientswith Crohn's disease can be classified, for example, into subtypes basedon the presence of fibrostenosing disease, internal-perforating disease,perianal fistulizing disease or ulcerative colitis-like diseaseaccording to previously described criteria. The fibrostenosing diseasesubtype is characterized by documented persistent intestinal obstructionor intestinal resection for intestinal obstruction. The extensive andoften protracted clinical testing required to diagnose Crohn's diseaseand disease subtypes may delay optimal treatment and involves invasiveprocedures such as endoscopy.

[0008] Identification of genetic markers which are closely associatedwith a clinical subtype of Crohn's disease would provide the basis fornovel genetic tests and eliminate or reduce the need for the battery oflaboratory, radiological, and endoscopic evaluations typically requiredto determine disease subtype. The availability of methods for diagnosingclinical subtypes of Crohn's disease would represent a major clinicaladvance that would aid in the therapeutic management of Crohn's diseaseand would further lay the groundwork for the design of treatmentmodalities which are specific to a particular disease subtype. Suchmethods can reduce costs associated with treatment of unresponsivedisease subtypes and eliminate the disappointment of those needlesslyundergoing ineffective therapy. In particular, a reliable genetic testfor the fibrostenosing subtype of Crohn's disease would be highly prizedas a non-invasive method for the early diagnosis of this disease subtypeand would also be useful for predicting susceptibility to thefibrostenosing subtype of Crohn's disease in asymptomatic individuals,making prophylactic therapy possible. The present invention satisfiesthis need and provides related advantages as well.

SUMMARY OF THE INVENTION

[0009] The present invention provides a method of diagnosing orpredicting susceptibility to a clinical subtype of Crohn's diseasecharacterized by fibrostenosing disease by determining the presence orabsence in an individual of a fibrostenosis-predisposing allele linkedto a NOD2/CARD15 locus, where the presence of thefibrostenosis-predisposing allele is diagnostic of or predictive ofsusceptibility to the clinical subtype of Crohn's disease characterizedby fibrostenosing disease. In a method of the invention, the clinicalsubtype of Crohn's disease can be, for example, characterized byfibrostenosing disease independent of small bowel involvement.

[0010] The invention also provides a method of optimizing therapy in anindividual by determining the presence or absence in the individual of afibrostenosis-predisposing allele linked to a NOD2/CARD15 locus,diagnosing individuals in which the fibrostenosis-predisposing allele ispresent as having a fibrostenosing subtype of Crohn's disease, andtreating the individual having a fibrostenosing subtype of Crohn'sdisease based on the diagnosis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows the NOD2/CARD15 locus intron and exon structure withthe location of SNP 8, SNP 12, SNP 13, and JW1 as well as other markers.

[0012]FIG. 2 shows the frequency of NOD2 variant carriers having atleast one of the three NOD2/CARD15 rare variant alleles (a “2” allele atSNP 8, SNP 12 or SNP 13) in Cohort 1 (“CD1”), Cohort 2 (“CD2”) or acombination of Cohort 1 and Cohort 2 (“Combined CD”). The striped barsindicate Crohn's disease patients with fibrostenosing disease and thesolid bars indicate Crohn's disease patients who did not have thefibrostenosing subtype of disease.

[0013]FIG. 3 shows the frequency of fibrostenosing complications inpatients relative to the number of NOD2/CARD15 rare variant alleles atSNP 8, SNP 12 or SNP 13. Based on genotyping at SNP 8, SNP 12 and SNP13, patients were described as carrying 0, 1, or 2 rare variant alleles(x axis, number of mutant NOD2 alleles). The left y axis shows thefrequency of fibrostenosing complications as filled circles. The right yaxis shows the odds ratio as a filled diamond with 95% confidenceintervals in parentheses. The * indicates a p value=0.008 and **indicates a p value of 0.004 compared with 0 alleles.

[0014]FIG. 4 shows a comparison of NOD2/CARD15 variant allelicfrequencies in patients with fibrostenosing disease compared withperforating disease. Patients were separated by the presence offibrostenosing disease with perforating complications (“Fib+perf”) orfibrostenosing disease without perforating complications (“Fib only”)compared with patients with perforating complications and withoutevidence of fibrostenosis (“Perf only”).

[0015]FIG. 5 shows the nucleotide sequence of NOD2/CARD15 surroundingSNP 8. The top strand is labeled as SEQ ID NO:1 and the bottom strand islabeled as SEQ ID NO:2. Nucleotide sequences which can be used asprimers for PCR amplification are indicated. In addition, the positionof a nucleotide sequence which can be used as a probe in an allelicdiscrimination assay is indicated, in this figure, by a box andlower-case letters. The underlined nucleotide indicates the position ofthe polymorphic site.

[0016]FIG. 6 shows the nucleotide sequence of NOD2/CARD15 surroundingSNP 12. The top strand is labeled as SEQ ID NO:3 and the bottom strandis labeled as SEQ ID NO:4. Nucleotide sequences which can be used asprimers for PCR amplification are indicated. In addition, the positionof a nucleotide sequence which can be used as a probe in an allelicdiscrimination assay is indicated, in this figure, by a box and lowercase letters. The underlined nucleotide indicates the position of thepolymorphic site.

[0017]FIG. 7 shows the nucleotide sequence of NOD2/CARD15 surroundingSNP 13. The top strand is labeled as SEQ ID NO:5 and the bottom strandis labeled as SEQ ID NO:6. Nucleotide sequences which can be used asprimers for PCR amplification are indicated. In addition, the positionof a nucleotide sequence which can be used as a probe in an allelicdiscrimination assay is indicated, in this figure, by a box and lowercase letters. The underlined nucleotide indicates the position of thepolymorphic site.

[0018]FIG. 8 shows the nucleotide sequence of NOD2/CARD15 surroundingSNP 5. The top strand is labeled as SEQ ID NO:7 and the bottom strand islabeled as SEQ ID NO:8. Nucleotide sequences which can be used asprimers for PCR amplification are indicated. In addition, the positionof a nucleotide sequence which can be used as a probe in an allelicdiscrimination assay is indicated, in this figure, by a box and lowercase letters. The underlined nucleotide indicates the position of thepolymorphic site.

[0019]FIG. 9 shows the nucleotide sequence of NOD2/CARD15 surroundingthe JW1 variant sequence. The top strand is labeled as SEQ ID NO:9 andthe bottom strand is labeled as SEQ ID NO:10. Nucleotide sequences whichcan be used as primers for PCR amplification are indicated. In addition,the position of a nucleotide sequence which can be used as a probe in anallelic discrimination assay is indicated, in this figure, by a box andlower case letters. The underlined nucleotide indicates the position ofthe polymorphic site.

[0020]FIG. 10 shows the nucleotide sequence of the 5′ untranslatedregion of NOD2/CARD15 in 12 individuals (SEQ ID NOS:12-23) compared tothe wild-type NOD2/CARD15 sequence (SEQ ID NO:11). Areas of sequenceidentity are shaded. The location of two polymorphic sites, JW18 andJW17, are indicated.

[0021]FIG. 11 shows the nucleotide sequence of the 3′ untranslatedregion of NOD2/CARD15 in 12 individuals. Areas of sequence identity areshaded. The location of two polymorphic sites, JW15 and JW16, areindicated. FIG. 11A shows the nucleotide sequence of the 3′ untranslatedregion of NOD2/CARD15 in 12 individuals (SEQ ID NOS:25-36) compared tothe wild-type NOD2/CARD15 sequence (SEQ ID NO:24) and the location ofJW16. FIG. 11B shows the nucleotide sequence of the 3′ untranslatedregion of NOD2/CARD15 in 12 individuals (SEQ ID NOS:56-67) compared tothe wild-type NOD2/CARD15 sequence (SEQ ID NO:55) and the location ofJW15.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention is directed to the exciting discovery ofdisease-predisposing alleles that are closely associated with thefibrostenosing disease subtype of Crohn's disease. Thesefibrostenosis-predisposing alleles are linked to a NOD2/CARD15 locus asdescribed further below and can be used to diagnose or predictsusceptibility to the fibrostenosing disease subtype of Crohn's disease.

[0023] As disclosed herein, genotyping and other clinicalcharacterization approaches were used to identify a strong associationbetween disease-predisposing alleles and the fibrostenosing diseasesubtype of Crohn's disease. In particular, two cohorts of Crohn'sdisease patients were assembled and clinically characterized (seeExample I and Table 1). These patients were also genotyped for threesingle nucleotide polymorphisms (SNPs) in the NOD2/CARD15 gene, SNP 8,SNP 12, and SNP 13, which are polymorphic markers associated withCrohn's disease. As disclosed herein, univariate analysis indicated thata “2” allele at SNP 8, SNP 12, or SNP 13 of the NOD2/CARD15 locus wassignificantly associated with fibrostenosing disease in Cohort 1(p=0.049, see Table 5). In addition, a positive association at a lessstringent significance level was also observed with small bowelinvolvement and younger age of onset, and a negative association wasobserved with ulcerative colitis-like disease in this cohort. Withrespect to serologic markers, patients with the “2” allele at SNP 13were more likely to express anti-Saccharomyces cerevisiae antibodies(ASCA) (p=0.053).

[0024] The results obtained with Cohort 1 were further tested usingCohort 2 as disclosed herein in Example IV. As with Cohort 1, Cohort 2demonstrated a significant association between a “2” allele at SNP 8,SNP 12, or SNP 13 of the NOD2/CARD15 locus and fibrostenosing disease(p=0.002, see Table 6). Furthermore, the significance between a “2”allele at SNP 8, SNP 12, or SNP 13 of the NOD2/CARD15 locus andfibrostenosing disease increased when the two cohorts were analyzedtogether (p=0.001, see FIG. 2).

[0025] As further disclosed herein in Example V, a “2” allele at SNP 8,SNP 12, or SNP 13 of the NOD2/CARD15 locus was associated withfibrostenosing disease in both Jewish and non-Jewish individuals.Approximately 46% of Crohn's disease patients with fibrostenosingdisease (Jewish individuals 52% vs. non-Jewish individuals 42%) had atleast one of these rare variant alleles compared with only 23% (Jewishindividuals 21.6% vs. non-Jewish individuals 25%) of Crohn's diseasepatients without fibrostenosing disease (Odds ratio, 2.8; 95% Confidenceinterval, 1.56-5.18). Of the three rare variant alleles, the “2” alleleat SNP 13, which is a frameshift mutation denoted “3020insC,”demonstrated the greatest association with fibrostenosing disease (47%vs. 17%, p=0.006 for cohorts combined). These results indicate thatfibrostenosis-predisposing alleles can be linked to the NOD2/CARD15locus.

[0026] As further disclosed herein in Example VI, patients who werecarriers of two fibrostenosis-predisposing alleles in NOD2/CARD15 weresignificantly more likely to have fibrostenosing disease as comparedwith patients who were not carriers of NOD2/CARD15 mutations at SNP 8,SNP 12 or SNP 13 (85% vs. 43%; odds ratio 7.4; 95% confidence interval1.9-28.9, p=0.004). See FIG. 3. Patients who were carriers of a singleNOD2/CARD15 fibrostenosis-predisposing allele were also significantlymore likely to have fibrostenosing disease when compared with patientswho were not carriers of any of the three NOD2/CARD15fibrostenosis-predisposing alleles assayed (64% vs. 43%; odds ratio2.37; 95% confidence interval 1.26-4.47; p=0.008). These results confirmthat patients who have a fibrostenosis-predisposing allele linked to aNOD2/CARD15 locus can have the fibrostenosing subtype of Crohn's diseaseand further indicate that Crohn's disease patients with multiplefibrostenosis-predisposing alleles (homozygous mutations or compoundheterozygous mutations) linked to NOD2/CARD15 have an increased risk offibrostenosing disease as compared to individuals carrying a singlefibrostenosis-predisposing allele.

[0027] Fibrostenosing and perforating disease can occur together in thesame patient. Patients with fibrostenosing disease can be characterized,for example, as i) having only fibrostenosing disease or ii) having bothfibrostenosing and perforating disease. As disclosed herein in ExampleVII, the percentage of patients having only fibrostenosing disease thatcarried a “2” allele at SNP 8, SNP 12, or SNP 13 of the NOD2/CARD15locus was 48.3%, which was similar to that seen in patients with bothfibrostenosing and perforating complications (46.0%; p=0.8). As seen inFIG. 4, when patients with fibrostenosing disease were compared withthose patients described as having perforating disease only (perianal orinternal), the frequency of the “2” allele at SNP 8, SNP 12 or SNP 13 ofthe NOD2/CARD15 locus in patients with fibrostenosing disease (with orwithout perforating complications) was significantly greater than thatseen in patients with only perforating complications (46.6% versus18.6%; p=0.002).

[0028] As further disclosed herein in Example VIII, multivariantanalysis was performed to investigate the association of a “2” allele atSNP 8, SNP 12, or SNP 13 of the NOD2/CARD15 locus with clinicalphenotypes. For multivariant analysis, all variables with at leastborderline significance (p<0.1) in either cohort were testedsimultaneously for their association with a “2” allele at SNP 8, SNP 12,or SNP 13 of the NOD2/CARD15 locus using logistic regression. As shownin Table 7, the clinical phenotype of fibrostenosing disease wassignificantly associated (p<0.05) with these rare alleles at theNOD2/CARD15 locus (odds ratio 2.8; 95% confidence interval, 1.3-6.0).These results confirm that fibrostenosing disease is independentlyassociated with a “2” allele at SNP 8, SNP 12, or SNP 13 of theNOD2/CARD15 locus.

[0029] Because fibrostenosing disease is more likely to occur inpatients with small-bowel involvement, patients were stratified on thebasis of small-bowel involvement to analyze whether the associationbetween fibrostenosing disease and NOD2/CARD15 variant alleles was aprimary association (see Example IX). Among patients with small-bowelinvolvement, 26.4% of patients who did not have fibrostenosing disease(n=53) had a “2” allele at SNP 8, SNP 12, or SNP 13, whereas 46.1% ofpatients who had fibrostenosing disease (n=102) had a “2” allele at SNP8, SNP 12, or SNP 13 (p=0.017). A similar trend was observed amongpatients without small-bowel involvement (p=0.05), and the combinedanalysis conditioning on small-bowel involvement yielded a significancelevel of 0.009. After controlling for fibrostenosing disease,small-bowel involvement was not associated with a “2” allele at SNP 8,SNP 12, or SNP 13 of the NOD2/CARD15 locus (p=0.63). This result agreeswith the results from logistic regression analysis (see Example VIII)and indicates that the association between fibrostenosing disease and a“2” allele at SNP 8, SNP 12, or SNP 13 of the NOD2/CARD15 locus isindependent of small-bowel involvement. These results further indicatethat the observed small-bowel association with a “2” allele at SNP 8,SNP 12, or SNP 13 of the NOD2/CARD15 locus is secondary to the presenceof fibrostenosing disease.

[0030] Based on these discoveries, the present invention provides amethod of diagnosing or predicting susceptibility to a clinical subtypeof Crohn's disease characterized by fibrostenosing disease bydetermining the presence or absence in an individual of afibrostenosis-predisposing allele linked to a NOD2/CARD15 locus, wherethe presence of the fibrostenosis-predisposing allele is diagnostic ofor predictive of susceptibility to the clinical subtype of Crohn'sdisease characterized by fibrostenosing disease. The methods of theinvention can be advantageous in that they are noninvasive and can beconveniently practiced, for example, with a blood sample from anindividual. The methods of the invention can be used to quickly, easilyand reliably diagnose or predict susceptibility to a clinical subtype ofCrohn's disease characterized by fibrostenosing disease as describedfurther herein below.

[0031] In one embodiment, a method of the invention is practiced with afibrostenosis-predisposing allele located within the NOD2/CARD15 locus.In another embodiment, NF-kappa B activation by a NOD2/CARD15polypeptide encoded by the fibrostenosis-predisposing allele is reducedas compared to NF-kappa B activation by a wild-type NOD2/CARD15polypeptide. In a further embodiment, a method of the invention ispracticed with a fibrostenosis-predisposing allele located in a codingregion of the NOD2/CARD15 locus, for example, within a region encodingresidues 744 to 1020 of NOD2/CARD15. In still a further embodiment, amethod of the invention is practiced with a fibrostenosis-predisposingallele which is a “2” allele at SNP 8, SNP 12, or SNP 13. In yet afurther embodiment, a method of the invention is practiced with afibrostenosis-predisposing allele which is a “2” allele at SNP 13. Inanother embodiment, a method of the invention is practiced with afibrostenosis-predisposing allele which is located in a non-codingregion of the NOD2/CARD15 locus. Such an allele can be, withoutlimitation, a JW1, JW15, or JW16 variant allele. In another embodiment,a method of the invention is practiced with a fibrostenosis-predisposingallele which is located in a promoter region of the NOD2/CARD15 locus.Useful fibrostenosis-predisposing alleles in the promoter region ofNOD2/CARD15 include, but are not limited to, JW17 and JW18 variantalleles.

[0032] The present invention relates to genetic markers which localizeto the IBD1 locus on chromosome 16. Utilizing genome wide scan linkagestrategies, the IBD1 locus was mapped to the proximal region of the longarm of chromosome 16 (16q12) in the Caucasian population (Hugot et al.,Nature 379:821-823 (1996)). This finding has been replicated in manystudies, including an international collaborative study reporting a highmultipoint linkage score (MLS) for a complex disease (MLS=5.7 at markerD16S411 in 16q12). See Cho et al., Inflamm. Bowel Dis. 3:186-190 (1997),Akolkar et al., Am. J. Gastroenterol. 96:1127-1132 (2001), Ohmen et al.,Hum. Mol. Genet. 5:1679-1683 (1996), Parkes et al., Lancet 348:1588(1996), Cavanaugh et al., Ann. Hum. Gent. (1998), Brant et al.,Gastroenteroloqy 115:1056-1061 (1998), Curran et al., Gastroenterlology115:1066-1071 (1998), Hampe et al., Am. J. Hum. Genet. 64:808-816(1999), and Annese et al., Eur. J. Hum. Genet. 7:567-573 (1999).NOD2/CARD15 within the IBD1 locus was simultaneously identified by apositional-cloning strategy (Hugot et al., Nature 411:599-603 (2001))and a positional candidate gene strategy (Ogura et al., Nature411:603-606 (2001), Hampe et al., Lancet 357:1925-1928 (2001)). Theencoded NOD2/CARD15 protein contains amino-terminal caspase recruitmentdomains (CARDs), which can activate NF-kappa B (NF-κB), and severalcarboxy-terminal leucine-rich repeat domains (Ogura et al, J. Biol.Chem. 276:4812-4818 (2001)). FIG. 1 shows an illustration of theNOD2/CARD15 locus.

[0033] The sequence of the human NOD2/CARD15 gene can be found inGenBank as accession number NM_(—)022162, which is incorporated byreference herein. In addition, the complete sequence of human chromosome16 clone RP11-327F22, which includes NOD2/CARD15, can be found inGenBank as accession number AC007728, which is incorporated by referenceherein. Furthermore, the sequence of NOD2/CARD15 from other species canbe found in the GenBank database.

[0034] Variations at three single nucleotide polymorphisms in the codingregion of NOD2/CARD15 have been previously described. These three SNPs,designated SNP 8, SNP 12 and SNP 13, are located in the carboxy-terminalregion of the NOD2/CARD15 gene (Hugot et al., supra, 2001).

[0035] The invention relies, in part, on determining the presence orabsence in an individual of a fibrostenosing-predisposing allele linkedto a NOD2/CARD15 locus. The term “fibrostenosis-predisposing allele,” asused herein, means a stably heritable molecular variation that is linkedto the NOD2/CARD15 locus and that tends to be inherited together withthe clinical subtype of Crohn's disease characterized by fibrostenosingdisease more often than would be expected according to traditionalMendelian genetics. A fibrostenosis-predisposing allele useful in theinvention can be, without limitation, a single nucleotide polymorphism(SNP), microsatellite (ms), variable number tandem repeat (VNTR)polymorphism, or a substitution, insertion or deletion of one or morenucleotides. One skilled in the art understands that afibrostenosis-predisposing allele also can be a molecular variation suchas abnormal methylation or other modification that does not produce adifference in the primary nucleotide sequence of thefibrostenosis-predisposing allele as compared to the wild type allele.

[0036] The term “linked to,” as used herein in reference to afibrostenosis-predisposing allele and a NOD2/CARD15 locus, means thatthe fibrostenosis-predisposing allele and the NOD2/CARD15 locus areinherited together more often than would be expected according totraditional Mendelian genetics. It is understood that an allele andlocus are linked when there is less than 50% recombination between thetwo sites. A fibrostenosis-predisposing allele is generally separatedfrom a NOD2/CARD15 locus by at most 50 centiMorgan (cM). As non-limitingexamples, a fibrostenosis-predisposing allele can be within 50centiMorgan (cM), 40 cM, 30 cM, 20 cM, 10 cM, 5 cM, or 1 cM of theNOD2/CARD15 locus. The distance between a linkedfibrostenosis-predisposing allele and a NOD2/CARD15 locus can also be,for example, 50,000,000 base pairs (bps), 40,000,000 bps, 30,000,000bps, 20,000,000 bps, 10,000,000 bps, 5,000,000 bps, 1,000,000 bps,500,000 bps, 100,000 bps, 50,000 bps, 10,000 bps, 1,000 bps or 100 bps.

[0037] The methods of the invention are useful for diagnosing orpredicting susceptibility to a clinical subtype of Crohn's disease(regional enteritis), which is a disease of chronic inflammation thatcan involve any part of the gastrointestinal tract. Commonly the distalportion of the small intestine (ileum) and cecum are affected in Crohn'sdisease. In other cases, the disease is confined to the small intestine,colon or anorectal region. Crohn's disease occasionally involves theduodenum and stomach, and more rarely the esophagus and oral cavity.

[0038] The variable clinical manifestations of Crohn's disease are, inpart, a result of the varying anatomic localization of the disease. Themost frequent symptoms of Crohn's disease are abdominal pain, diarrheaand recurrent fever. Crohn's disease is commonly associated withintestinal obstruction or fistula, which is an abnormal passage, forexample, between diseased loops of bowel. Crohn's disease also caninclude extra-intestinal complications such as inflammation of the eye,joints and skin; liver disease; kidney stones or amyloidosis; and isassociated with an increased risk of intestinal cancer.

[0039] Several features are characteristic of the pathology of Crohn'sdisease. The inflammation associated with Crohn's disease, known astransmural inflammation, involves all layers of the bowel wall.Thickening and edema, for example, typically appear throughout the bowelwall, with fibrosis also present in long-standing disease. Theinflammation characteristic of Crohn's disease also is discontinuous,with segments of inflamed tissue, known as “skip lesions,” separated byapparently normal intestine. Furthermore, linear ulcerations, edema, andinflammation of the intervening tissue lead to a “cobblestone”appearance of the intestinal mucosa, which is distinctive of Crohn'sdisease.

[0040] A hallmark of Crohn's disease is the presence of discreteaggregations of inflammatory cells, known as granulomas, which aregenerally found in the submucosa. About half of Crohn's disease casesdisplay the typical discrete granulomas, while others show a diffusegranulomatous reaction or nonspecific transmural inflammation. As aresult, the presence of discrete granulomas is indicative of Crohn'sdisease, although the absence granulomas also is consistent with thedisease. Thus, transmural or discontinuous inflammation, rather than thepresence of granulomas, is a preferred diagnostic indicator of Crohn'sdisease (Rubin and Farber, Pathology (Second Edition) Philadelphia: J.B. Lippincott Company (1994)).

[0041] Crohn's disease is a classification representing a number ofheterogeneous disease subtypes that affect the gastrointestinal tractand may produce similar symptoms. As non-limiting examples, patientswith Crohn's disease can be characterized as having subtypescharacterized by fibrostenosing disease, internal-perforating disease,perianal fistulizing disease or ulcerative colitis (UC)-like diseasebased on previously described criteria (Gasche et al., InflammatoryBowel Diseases 6:8-15 (2000); Vasiliauskas et al., Gut 47:487-496(2000); Vasiliauskas et al., Gastroenterology 110:1810-1819 (1996); andGreenstein et al., Gut 29:588-592 (1988)).

[0042] According to well-established criteria fibrostenosing disease isdefined by documented persistent intestinal obstruction or an intestinalresection for an intestinal obstruction. Radiographic, endoscopic,surgical or histopathological documentation can be used to confirm adiagnosis of the fibrostenosing subtype of Crohn's disease. Thefibrostenosing subtype of Crohn's disease can be accompanied by othersymptoms such as perforations, abscesses or fistulae. In addition, thefibrostenosing subtype of Crohn's disease can be characterized bypersistent symptoms of intestinal blockage such as nausea, vomiting,abdominal distention and inability to eat solid food. Intestinal X-raysof patients with the fibrostenosing subtype of Crohn's disease can show,for example, distention of the bowel before the point of blockage.

[0043] Additional subtypes of Crohn's disease can be identified usingdefined clinical criteria. For example, internal perforating disease canbe defined as current or previous evidence of entero-enteric orentero-vesicular fistulae, intraabdominal abscesses, or small bowelperforation. Perianal perforating disease can be defined by current orprevious evidence of either perianal fistulae or abscesses orrectovaginal fistula. UC-like disease can be defined by current orprevious evidence of left-sided colonic involvement, symptoms ofbleeding or urgency, and crypt abscesses on colonic biopsies aspreviously described. Disease location can be classified as small bowel,colon, or both based on one or more endoscopic, radiologic or pathologicstudies.

[0044] The fibrostenosing subtype of Crohn's disease can occur inpatients having disease with small-bowel involvement. As disclosedherein, after controlling for fibrostenosing disease, small-bowelinvolvement was not associated with a “2” allele at SNP 8, SNP 12, orSNP 13 of the NOD2/CARD15 locus (p=0.63; see Example IX). This resultagrees with the results from logistic regression analysis disclosed inExample VIII and indicates that the association between fibrostenosingdisease and a “2” allele at SNP 8, SNP 12, or SNP 13 of the NOD2/CARD15locus is independent of small-bowel involvement. Based on thesefindings, the present invention provides a method of diagnosing orpredicting susceptibility to a clinical subtype of Crohn's diseasecharacterized by fibrostenosing disease independent of small bowelinvolvement by determining the presence or absence in an individual of afibrostenosis-predisposing allele linked to a NOD2/CARD15 locus, wherethe presence of the fibrostenosis-predisposing allele is diagnostic ofor predictive of susceptibility to the clinical subtype of Crohn'sdisease characterized by fibrostenosing disease independent of smallbowel involvement.

[0045] The diagnostic methods of the invention are practiced in anindividual. As used herein, the term “individual” means an animal, suchas a human or other mammal, capable of having the fibrostenosing subtypeof Crohn's disease. An individual can have one or more symptoms ofCrohn's disease or the fibrostenosing subtype of Crohn's disease or canbe asymptomatic. The methods of the invention can be useful, forexample, for diagnosing the fibrostenosing subtype of Crohn's disease inan individual with one or more symptoms, or for predictingsusceptibility to the fibrostenosing subtype of Crohn's disease in anasymptomatic individual such as an individual at increased risk forhaving the fibrostenosing subtype of Crohn's disease. In one embodiment,the methods of the invention are used to determine the presence orabsence of the fibrostenosing subtype of Crohn's disease in anindividual known to have Crohn's disease.

[0046] The methods of the invention can be useful, for example, todiagnose or predict susceptibility to the fibrostenosing subtype ofCrohn's disease in an Ashkenazi Jewish individual. Crohn's disease issignificantly more common (2 to 8 fold higher) in Ashkenazi Jews than innon-Jewish Caucasians (Brant et al., Gastroenterol. 115:1056-1061(1998)). Furthermore, among persons of Jewish ethnicity, American orEuropean Ashkenazi Jews have a 2 to 4 fold increased risk of having thisinflammatory bowel disease compared with Sephardic or Oriental Jews(Yang and Rotter in Kirschner and Shorter (Eds.), Inflammatory BowelDisease Baltimore: Williams and Wilkins, p. 301-331 (1995); Rozen etal., Gastroenterol. 76:25-30 (1979)). The empiric risk of Crohn'sdisease for a first degree relative of a proband with Crohn's disease is7.8% for Jews compared with 5.2% for non-Jews (p=0.005; Yang et al., Gut34:517-524 (1993)). Thus, the Jewish population and especially theAshkenazi Jewish population represents a group at increased risk forCrohn's and autoimmune diseases of related etiology.

[0047] The methods of the invention rely on fibrostenosis-predisposingalleles linked to the NOD2/CARD15 locus. NOD2/CARD15 has structuralhomology with both apoptosis regulators Apaf-1/CED-4 and a class ofplant disease resistant gene products (Ogura et al., J. Biol. Chem,276:4812-4818 (2001)). Similar to plant disease resitant gene products,NOD2/CARD15 has an amino-terminal effector domain, a nucleotide-bindingdomain and leucine rich repeats (LRRs). Wild-type NOD2/CARD15 activatesnuclear factor NF-kappa B, making it responsive to bacteriallipopolysaccharides (LPS; Ogura et al., J. Biol. Chem, 276:4812-4818(2001); Inohara et al., J. Biol. Chem. 276:2551-2554 (2001). NOD2/CARD15can function as an intercellular receptor for LPS, with the leucine richrepeats required for responsiveness. Like NOD2/CARD15, cytosolic plantdisease resistant polypeptides have carboxy-terminal leucine richrepeats that are important for the recognition of pathogen componentsand induction of pathogen-specific responses (Parniske et al., Cell91:821-832 (1997); Ellis et al., Plant Cell 11:495-506 (1999); Dixon etal., Proc. Natl. Acad. Sci. USA 97: 8807-8814 (2000)).

[0048] In one embodiment, the fibrostenosis-predisposing allele islocated within the NOD2/CARD15 locus, a schematic of which is shown inFIG. 1. The NOD2/CARD15 locus includes coding regions of the NOD2/CARD15gene as well as non-coding regions such as introns and 5′ and 3′untranslated regions. One skilled in the art understands that theNOD2/CARD15 locus can include, for example, promoter regions 5′ of thegene, enhancer regions 5′ or 3′ of the gene or in intronic sequences,and mRNA stability regions 3′ of the gene.

[0049] In another embodiment, the fibrostenosis-predisposing allele islocated in a coding region of the NOD2/CARD15 locus, for example withina region encoding residues 744 to 1020 of NOD2/CARD15. Residues 744 to1020 of the NOD2/CARD15 polypeptide contain several leucine-rich repeatsin the carboxy-terminal portion of the NOD2/CARD15 polypeptide.Fibrostenosis-predisposing alleles located in a region encoding residues744 to 1020 of NOD2/CARD15 include, without limitation, SNP 12 and SNP13. A fibrostenosis-predisposing allele useful in the invention also canbe an allele which encodes a NOD2/CARD15 polypeptide with reducedNF-kappa B activation as compared to NF-kappa B activation by awild-type NOD2/CARD15 polypeptide. As an example, a rare variant alleleat SNP 13 results in a truncated NOD2/CARD15 polypeptide which hasreduced ability to induce NF-kappa B in response to LPS stimulation(Ogura et al., Nature 411:603-606 (2001)).

[0050] A fibrostenosis-predisposing allele useful in the invention canbe, for example, a “2” allele at SNP 8, SNP 12 or SNP 13. SNP 8, SNP 12,and SNP 13 are located within the coding region of NOD2/CARD15 as shownin FIG. 1. In one embodiment, a method of the invention is practicedwith a fibrostenosis-predisposing allele which is the SNP 8 “2” allele.As used herein, the term “SNP 8” means a single nucleotide polymorphismwithin exon 4 in the NOD2/CARD15 gene, which occurs within a tripletencoding amino acid 702 of the NOD2/CARD15 protein. The “1” allele, inwhich cytosine (c) resides at position 138,991 of the AC007728 sequence,is the common or “wild-type” SNP 8 allele and occurs within a tripletencoding an arginine at amino acid 702. The “2” allele of SNP 8, inwhich thymine (t) resides at position 138,991 of the AC007728 sequence,is a rare variant that results in an arginine (R) to tryptophan (W)substitution at amino acid 702 of the NOD2/CARD15 protein. Accordingly,the rare “2” allele at SNP 8 is denoted “R702W” or “702W” and can alsobe denoted “R675W” based on the earlier numbering system of Hugot etal., supra, 2001. The NCBI SNP ID number for SNP 8 is rs2066844, whichis incorporated herein by reference. As disclosed herein and describedfurther below, the presence of allele “1” or “2” at SNP 8, or anotherSNP described below, can be conveniently detected, for example, byallelic discrimination assays or sequence analysis.

[0051] A method of the invention also can be practiced with afibrostenosis-predisposing allele which is the SNP 12 “2” allele. Asused herein, the term “SNP 12” means a single nucleotide polymorphismwithin exon 8 in the NOD2/CARD15 gene, which occurs within a tripletencoding amino acid 908 of the NOD2/CARD15 protein. The “1” allele, inwhich guanine (g) resides at position 128,377 of the AC007728 sequence,is the common or “wild-type” SNP 12 allele and occurs within a tripletencoding glycine at amino acid 908. The “2” allele of SNP 12, in whichcytosine (c) resides at position 128,377 of the AC007728 sequence, is arare variant that results in a glycine (G) to arginine (R) substitutionat amino acid 908 of the NOD2/CARD15 protein. This rare “2” allele atSNP 12 is denoted “G908R” or “908R” and can also be denoted “G881R”based on the earlier numbering system of Hugot et al., supra, 2001. SNP12 is located within the leucine rich repeat region of the NOD2/CARD15gene. The NCBI SNP ID number for SNP 12 is rs2066845, which isincorporated herein by reference.

[0052] A method of the invention also can be practiced with afibrostenosis-predisposing allele which is the SNP 13 “2” allele. Thisallele is an insertion of a single nucleotide that results in a frameshift in the tenth leucine-rich repeat of the NOD2/CARD15 protein and isfollowed by a premature stop codon. The resulting truncation of theNOD2/CARD15 protein appears to prevent activation of NF-κB in responseto bacterial lipopolysaccharides (Ogura et al., supra, 2001). As usedherein, the term “SNP 13” means a single nucleotide polymorphism withinexon 11 in the NOD2/CARD15 gene, which occurs in a triplet encodingamino acid 1007 of the NOD2/CARD15 protein. The “2” allele of SNP 13, inwhich a cytosine has been added at position 121,139 of the AC007728sequence, is a rare variant resulting in a frame shift mutation at aminoacid 1007. Accordingly, the rare “2” allele at SNP 13 is denoted“1007fs” and can also be denoted “3020insC,” or “980fs” based on theearlier numbering system of Hugot et al., supra, 2001. The NCBI SNP IDnumber for SNP 13 is rs2066847, which is incorporated herein byreference.

[0053] One skilled in the art recognizes that a particular polymorphicallele can be conveniently defined, for example, in comparison to aCentre d′Etude du Polymorphisme Humain (CEPH) reference individual suchas the individual designated 1347-02 (Dib et al., Nature 380:152-154(1996)), using commercially available reference DNA obtained, forexample, from PE Biosystems (Foster City, Calif.). In addition, specificinformation on SNPs can be obtained from the dbSNP of the NationalCenter for Biotechnology Information (NCBI).

[0054] A fibrostenosis-predisposing allele also can be located in anon-coding region of the NOD2/CARD15 locus. Non-coding regions include,for example, intron sequences as well as 5′ and 3′ untranslatedsequences. Examples of a fibrostenosis-predisposing allele in an intronsequence of the NOD2/CARD15 gene include, but are not limited to, theJW1 variant allele, which is described below. Examples offibrostenosis-predisposing alleles located in the 3′ untranslated regionof the NOD2/CARD15 gene include, without limitation, JW15 and JW16variant alleles, which are described below. Examples offibrostenosis-predisposing alleles located in the 5′ untranslated regionof the NOD2/CARD15 gene include, without limitation, the JW17 and JW18variant alleles, which are described below. In one embodiment, a methodof the invention is practiced with a fibrostenosis-predisposing allelelocated in a non-coding region of the NOD2/CARD15 locus which is a JW1,JW15 or JW16 variant allele. In another embodiment, a method of theinvention is practiced with a fibrostenosis-predisposing allele locatedin a promoter region of the NOD2/CARD15 locus which is a JW17 or JW18variant allele.

[0055] As used herein, the term “JW1 variant allele” means a geneticvariation at nucleotide 158 of intervening sequence 8 (intron 8) of aNOD2/CARD15 gene. In relation to the AC007728 sequence, the JW1 variantis located at position 128,143. The genetic variation at nucleotide 158of intron 8 can be, but is not limited to, a single nucleotidesubstitution, multiple nucleotide substitutions, or a deletion orinsertion of one or more nucleotides. The wild type sequence of intron 8has a cytosine at position 158; as non-limiting examples, a JW1 variantallele can have a cytosine (C) to adenine (A), cytosine to guanine (G),or cytosine to thymine (T) substitution at nucleotide 158 of intron 8.In one embodiment, the JW1 variant allele is a change from a cytosine toa thymine at nucleotide 158 of NOD2/CARD15 intron 8.

[0056] As used herein, the term “JW15 variant allele” means a geneticvariation in the 3′ untranslated region of NOD2/CARD15 at nucleotideposition 118,790 of the AC007728 sequence. The genetic variation atnucleotide 118,790 can be, but is not limited to, a single nucleotidesubstitution, multiple nucleotide substitutions, or a deletion orinsertion of one or more nucleotides. The wild type sequence has anadenine (A) at position 118,790; as non-limiting examples, a JW15variant allele can have an adenine (A) to cytosine (C), adenine toguanine (G), or adenine to thymine (T) substitution at nucleotide118,790. In one embodiment, the JW15 variant allele is a change from anadenine to a cytosine at nucleotide 118,790.

[0057] As used herein, the term “JW16 variant allele” means a geneticvariation in the 3′ untranslated region of NOD2/CARD15 at nucleotideposition 118,031 of the AC007728 sequence. The genetic variation atnucleotide 118,031 can be, but is not limited to, a single nucleotidesubstitution, multiple nucleotide substitutions, or a deletion orinsertion of one or more nucleotides. The wild type sequence has aguanine (G) at position 118,031; as non-limiting examples, a JW16variant allele can have a guanine (G) to cytosine (C), guanine toadenine (A), or guanine to thymine (T) substitution at nucleotide118,031. In one embodiment, the JW16 variant allele is a change from aguanine to an adenine at nucleotide 118,031.

[0058] As used herein, the term “JW17 variant allele” means a geneticvariation in the 5′ untranslated region of NOD2/CARD15 at nucleotideposition 154,688 of the AC007728 sequence. The genetic variation atnucleotide 154,688 can be, but is not limited to, a single nucleotidesubstitution, multiple nucleotide substitutions, or a deletion orinsertion of one or more nucleotides. The wild type sequence has acytosine (C) at position 154,688; as non-limiting examples, a JW17variant allele can have a cytosine (C) to guanine (G), cytosine toadenine (A), or cytosine to thymine (T) substitution at nucleotide154,688. In one embodiment, the JW17 variant allele is a change from acytosine to a thymine at nucleotide 154,688.

[0059] As used herein, the term “JW18 variant allele” means a geneticvariation in the 5′ untranslated region of NOD2/CARD15 at nucleotideposition 154,471 of the AC007728 sequence. The genetic variation atnucleotide 154,471 can be, but is not limited to, a single nucleotidesubstitution, multiple nucleotide substitutions, or a deletion orinsertion of one or more nucleotides. The wild type sequence has acytosine (C) at position 154,471; as non-limiting examples, a JW18variant allele can have a cytosine (C) to guanine (G), cytosine toadenine (A), or cytosine to thymine (T) substitution at nucleotide154,471. In one embodiment, the JW18 variant allele is a change from acytosine to a thymine at nucleotide 154,471.

[0060] Further provided herein is a method of diagnosing or predictingsusceptibility to a clinical subtype of Crohn's disease characterized byfibrostenosing disease by determining the presence or absence in anindividual of at least two fibrostenosis-predisposing alleles linked toa NOD2/CARD15 locus, where the presence of one or more of thefibrostenosis-predisposing alleles is diagnostic of or predictive ofsusceptibility to the clinical subtype of Crohn's disease characterizedby fibrostenosing disease. In a method of the invention, the at leasttwo fibrostenosis-predisposing alleles are “2” alleles at SNP 8, SNP 12or SNP 13.

[0061] Further provided herein is a method of diagnosing or predictingsusceptibility to a clinical subtype of Crohn's disease characterized byfibrostenosing disease by determining the presence or absence in anindividual of at least two fibrostenosis-predisposing alleles linked toa NOD2/CARD15 locus, where the presence of one or more of thefibrostenosis-predisposing alleles is diagnostic of or predictive ofsusceptibility to the clinical subtype of Crohn's disease characterizedby fibrostenosing disease. In one embodiment, the at least twofibrostenosis-predisposing alleles are “2” alleles at SNP 8, SNP 12, orSNP 13. In another embodiment, a method of the invention for diagnosingor predicting susceptibility to a clinical subtype of Crohn's diseasecharacterized by fibrostenosing disease is practiced by determining thepresence or absence in the individual of (i) a “2” allele at SNP 8, (ii)a “2” allele at SNP 12, and (iii) a “2” allele at SNP 13, where thepresence of one or more of the “2” alleles at SNP 8, SNP 12, and SNP 13is diagnostic of or predictive of susceptibility to the clinical subtypeof Crohn's disease characterized by fibrostenosing disease.

[0062] The strength of the association between afibrostenosing-predisposing allele and Crohn's disease can becharacterized by a particular odds ratio such as an odds ratio of atleast 2 with a lower 95% confidence interval limit of greater than 1.Such an odds ratio can be, for example, at least 3.0, 4.0, 5.0, 6.0,7.0, or 8.0 or greater with a lower 95% confidence interval limit ofgreater than 1, such as an odds ratio of at least 7.4 with a 95%confidence interval of 1.9-28.9 (see FIG. 3). In addition, an odds ratiocan be, for example, at least 2.37 with a lower confidence intervallimit of 1.26-4.47 (see FIG. 3). In one embodiment, thefibrostenosis-predisposing allele is associated with the clinicalsubtype of Crohn's disease characterized by fibrostenosing disease withan odds ratio of at least 2 and a lower 95% confidence limit greaterthan 1. Methods for determining an odds ratio are well known in the art(see, for example, Schlesselman et al., Case Control Studies: Design,Conduct and Analysis Oxford University Press, New York (1982)).

[0063] In one embodiment, a fibrostenosis-predisposing allele isassociated with the fibrostenosing subtype of Crohn's disease with a pvalue of equal to or less than 0.05. In other embodiments, afibrostenosis-predisposing allele is associated with the fibrostenosingsubtype of Crohn's disease with a p value of equal to or less than 0.1.As used herein, the term “p value” is synonymous with “probabilityvalue.” As is well known in the art, the expected p value for theassociation between a random allele and disease is 1.00. A p value ofless than about 0.05 indicates that the allele and disease do not appeartogether by chance but are influenced by positive factors. Generally,the statistical threshold for significance of linkage has been set at alevel of allele sharing for which false positives would occur once intwenty genome scans (p=0.05). In particular embodiments, afibrostenosis-predisposing allele is associated with a clinical subtypeof Crohn's disease characterized by fibrostenosis with a p value ofequal to or less than 0.1, 0.05, 0.04, 0.03, 0.02, 0.01, 0.009, 0.008,0.007, 0.006, 0.005, 0.004, 0.003, 0.002 or 0.001, or with a p value ofless than 0.00095, 0.0009, 0.00085, 0.0008 or 0.0005. It is recognizedthat, in some cases, p values may need to be corrected, for example, toaccount for factors such as sample size (number of families), geneticheterogeneity, clinical heterogeneity, or analytical approach(parametric or nonparametric method).

[0064] A variety of means can be used to determine the presence orabsence of a fibrostenosing-predisposing allele in a method of theinvention. As an example, enzymatic amplification of nucleic acid froman individual can be conveniently used to obtain nucleic acid forsubsequent analysis. The presence or absence of afibrostenosis-predisposing allele also can be determined directly fromthe individual's nucleic acid without enzymatic amplification.

[0065] Analysis of nucleic acid from an individual, whether amplified ornot, can be performed using any of various techniques. Useful techniquesinclude, without limitation, polymerase chain reaction based analysis,sequence analysis and electrophoretic analysis, which can be used aloneor in combination. As used herein, the term “nucleic acid” means apolynucleotide such as a single- or double-stranded DNA or RNA moleculeincluding, for example, genomic DNA, cDNA and mRNA. The term nucleicacid encompasses nucleic acid molecules of both natural and syntheticorigin as well as molecules of linear, circular or branchedconfiguration representing either the sense or antisense strand, orboth, of a native nucleic acid molecule. It is understood that suchnucleic acid can be attached to a synthetic material such as a bead orcolumn matrix.

[0066] The presence or absence of a fibrostenosing-predisposing allelecan involve amplification of an individual's nucleic acid by thepolymerase chain reaction. Use of the polymerase chain reaction for theamplification of nucleic acids is well known in the art (see, forexample, Mullis et al. (Eds.), The Polymerase Chain Reaction,Birkhauser, Boston, (1994)). In one embodiment, polymerase chainreaction amplification is performed using one or more fluorescentlylabeled primers. In another embodiment, polymerase chain reactionamplification is performed using one or more labeled or unlabeledprimers that contain a DNA minor grove binder.

[0067] Several different primers can be used to amplify an individual'snucleic acid by the polymerase chain reaction. For example, the PCRprimers listed in Table 2 (SEQ ID NOS: 37-44) and shown in FIGS. 5 to 8can be used to amplify specific regions in the NOD2/CARD15 locus of anindividual's nucleic acid. For example, the region surrounding SNP 8 canbe amplified using SEQ ID NO: 39 and 40; SNP 12 can be amplified usingSEQ ID NOS: 41 and 42, and the region surrounding SNP 13 can beamplified using SEQ ID NOS: 43 and 44. In addition, for example, theregion surrounding. As understood by one skilled in the art, additionalprimers for PCR analysis can be designed based on the sequence flankingthe region of interest. As a non-limiting example, a sequence primer cancontain about 15 to 30 nucleotides of a sequence upstream or downstreamof the region of interest. Such primers are generally designed to havesufficient guanine and cytosine content to attain a high meltingtemperature which allows for a stable annealing step in theamplification reaction. Several computer programs, such as PrimerSelect, are available to aid in the design of PCR primers.

[0068] A Taqman® allelic discrimination assay available from AppliedBiosystems can be useful for determining the presence or absence of afibrostenosing-predisposing allele. In a Taqman® allelic discriminationassay, a specific, fluorescent, dye-labeled probe for each allele isconstructed. The probes contain different fluorescent reporter dyes suchas FAM and VICTM to differentiate the amplification of each allele. Inaddition, each probe has a quencher dye at one end which quenchesfluorescence by fluorescence resonance energy transfer (FRET). DuringPCR, each probe anneals specifically to complementary sequences in thenucleic acid from the individual. The 5′ nuclease activity of Taqpolymerase is used to cleave only probe that hybridize to the allele.Cleavage separates the reporter dye from the quencher dye, resulting inincreased fluorescence by the reporter dye. Thus, the fluorescencesignal generated by PCR amplification indicates which alleles arepresent in the sample. Mismatches between a probe and allele reduce theefficiency of both probe hybridization and cleavage by Taq polymerase,resulting in little to no fluorescent signal. Improved specificity inallelic discrimination assays can be achieved by conjugating a DNA minorgrove binder (MGB) group to a DNA probe as described, for example, inKutyavin et al., Nuc. Acids Research 28:655-661 (2000). Minor grovebinders include, but are not limited to, compounds such asdihydrocyclopyrroloindole tripeptide (DPI3).

[0069] Sequence analysis also can be useful for determining the presenceor absence of a fibrostenosing-predisposing allele in a method of theinvention. A fibrostenosing-predisposing allele can be detected bysequence analysis using primers disclosed herein, for example, as thePCR primers listed in Table 2 (SEQ ID NOS: 37-44) and shown in FIGS. 5to 8. As understood by one skilled in the art, additional primers forsequence analysis can be designed based on the sequence flanking theregion of interest. As a non-limiting example, a sequence primer cancontain about 15 to 30 nucleotides of a sequence about 40 to 400 basepairs upstream or downstream of the region of interest. Such primers aregenerally designed to have sufficient guanine and cytosine content toattain a high melting temperature which allows for a stable annealingstep in the sequencing reaction.

[0070] The term “sequence analysis,” as used herein in reference to oneor more nucleic acids, means any manual or automated process by whichthe order of nucleotides in the nucleic acid is determined. As anexample, sequence analysis can be used to determine the nucleotidesequence of a sample of DNA. The term sequence analysis encompasses,without limitation, chemical and enzymatic methods such as dideoxyenzymatic methods including, for example, Maxam-Gilbert and Sangersequencing as well as variations thereof. The term sequence analysisfurther encompasses, but is not limited to, capillary array DNAsequencing, which relies on capillary electrophoresis and laser-inducedfluorescence detection and can be performed using instruments such asthe MegaBACE 1000 or ABI 3700. As additional non-limiting examples, theterm sequence analysis encompasses thermal cycle sequencing (Sears etal., Biotechniques 13:626-633 (1992)); solid-phase sequencing (Zimmermanet al., Methods Mol. Cell Biol. 3:39-42 (1992); and sequencing with massspectrometry such as matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry MALDI-TOF MS (Fu et al., NatureBiotech. 16: 381-384 (1998)). The term sequence analysis also includes,yet is not limited to, sequencing by hybridization (SBH), which relieson an array of all possible short oligonucleotides to identify a segmentof sequences present in an unknown DNA (Chee et al., Science 274:610-614(1996); Drmanac et al., Science 260:1649-1652 (1993); and Drmanac etal., Nature Biotech. 16:54-58 (1998)). One skilled in the artunderstands that these and additional variations are encompassed by theterm sequence analysis as defined herein. See, in general, Ausubel etal., supra, Chapter 7 and supplement 47.

[0071] The invention also provides a method of diagnosing or predictingsusceptibility to a clinical subtype of Crohn's disease characterized byfibrostenosing disease by determining the presence or absence in anindividual of a fibrostenosis-predisposing allele linked to aNOD2/CARD15 locus, where the presence of the fibrostenosis-predisposingallele is diagnostic of or predictive of susceptibility to the clinicalsubtype of Crohn's disease characterized by fibrostenosing disease, andwhere the method includes the steps of obtaining material containingnucleic acid including the NOD2/CARD15 locus from the individual. Asused herein, the term “material” means any biological matter from whichnucleic acid can be prepared. As non-limiting examples, the termmaterial encompasses whole blood, plasma, saliva, cheek swab, or otherbodily fluid or tissue that contains nucleic acid. In one embodiment, amethod of the invention is practiced with whole blood, which can beobtained readily by non-invasive means and used to prepare genomic DNA,for example, for enzymatic amplification or automated sequencing. Inanother embodiment, a method of the invention is practiced with tissueobtained from an individual such as tissue obtained during surgery orbiopsy procedures.

[0072] Electrophoretic analysis also can be useful in the methods of theinvention. Elecrophoretic analysis, as used herein in reference to oneor more nucleic acids such as amplified fragments, means a processwhereby charged molecules are moved through a stationary medium underthe influence of an electric field. Electrophoretic migration separatesnucleic acids primarily on the basis of their charge, which is inproportion to their size, with smaller molecules migrating more quickly.The term electrophoretic analysis includes, without limitation, analysisusing slab gel electrophoresis, such as agarose or polyacrylamide gelelectrophoresis, or capillary electrophoresis. Capillary electrophoreticanalysis generally occurs inside a small-diameter (50-100-m) quartzcapillary in the presence of high (kilovolt-level) separating voltageswith separation times of a few minutes. Using capillary electrophoreticanalysis, nucleic acids are conveniently detected by UV absorption orfluorescent labeling, and single-base resolution can be obtained onfragments up to several hundred base pairs. Such methods ofelectrophoretic analysis, and variations thereof, are well known in theart, as described, for example, in Ausubel et al., Current Protocols inMolecular Bioloqy Chapter 2 (Supplement 45) John Wiley & Sons, Inc. NewYork (1999).

[0073] Restriction fragment length polymorphism (RFLP) analysis also canbe useful for determining the presence or absence of afibrostenosis-predisposing allele in a method of the invention (Jarchoet al. in Dracopoli et al., Current Protocols in Human Genetics pages2.7.1-2.7.5, John Wiley & Sons, New York; Innis et al.,(Ed.), PCRProtocols, San Diego: Academic Press, Inc. (1990)). As used herein,restriction fragment length polymorphism analysis is any method fordistinguishing genetic polymorphisms using a restriction enzyme, whichis an endonuclease that catalyzes the degradation of nucleic acid andrecognizes a specific base sequence, generally a palindrome or invertedrepeat. One skilled in the art understands that the use of RFLP analysisdepends upon an enzyme that can differentiate two alleles at apolymorphic site.

[0074] Allele-specific oligonucleotide hybridization also can be used todetect the presence or absence of a fibrostenosis-predisposing allele.Allele-specific oligonucleotide hybridization is based on the use of alabeled oligonucleotide probe having a sequence perfectly complementary,for example, to the sequence encompassing a fibrostenosis-predisposingallele. Under appropriate conditions, the allele-specific probehybridizes to a nucleic acid containing the fibrostenosis-predisposingallele but does not hybridize to the one or more other alleles, whichhave one or more nucleotide mismatches as compared to the probe. Ifdesired, a second allele-specific oligonucleotide probe that matches analternate allele also can be used. Similarly, the technique ofallele-specific oligonucleotide amplification can be used to selectivelyamplify, for example, a fibrostenosis-predisposing allele by using anallele-specific oligonucleotide primer that is perfectly complementaryto the nucleotide sequence of the fibrostenosis-predisposing allele butwhich has one or more mismatches as compared to other alleles (Mullis etal., supra, 1994). One skilled in the art understands that the one ormore nucleotide mismatches that distinguish between thefibrostenosis-predisposing allele and one or more other alleles areoften located in the center of an allele-specific oligonucleotide primerto be used in allele-specific oligonucleotide hybridization. Incontrast, an allele-specific oligonucleotide primer to be used in PCRamplification generally contains the one or more nucleotide mismatchesthat distinguish between the disease-associated and other alleles at the3′ end of the primer.

[0075] A heteroduplex mobility assay (HMA) is another well known assaythat can be used to detect the presence or absence of afibrostenosis-predisposing allele in a method of the invention. HMA isuseful for detecting the presence of a polymorphic sequence since a DNAduplex carrying a mismatch has reduced mobility in a polyacrylamide gelcompared to the mobility of a perfectly base-paired duplex (Delwart etal., Science 262:1257-1261 (1993); White et al., Genomics 12:301-306(1992)).

[0076] The technique of single strand conformational polymorphism (SSCP)also can be used to detect the presence or absence of afibrostenosis-predisposing allele in a method of the invention (seeHayashi, Methods Applic. 1:34-38 (1991)). This technique is used todetect mutations based on differences in the secondary structure ofsingle-strand DNA that produce an altered electrophoretic mobility uponnon-denaturing gel electrophoresis. Polymorphic fragments are detectedby comparison of the electrophoretic pattern of the test fragment tocorresponding standard fragments containing known alleles.

[0077] Denaturing gradient gel electrophoresis (DGGE) also can be usedto detect a fibrostenosis-predisposing allele in a method of theinvention. In DGGE, double-stranded DNA is electrophoresed in a gelcontaining an increasing concentration of denaturant; double-strandedfragments made up of mismatched alleles have segments that melt morerapidly, causing such fragments to migrate differently as compared toperfectly complementary sequences (Sheffield et al., “Identifying DNAPolymorphisms by Denaturing Gradient Gel Electrophoresis” in Innis etal., supra, 1990).

[0078] Other molecular methods useful for determining the presence orabsence of a fibrostenosis-predisposing allele are known in the art anduseful in the methods of the invention. Other well-known approaches fordetermining the presence or absence of a fibrostenosis-predisposingallele include, without limitation, automated sequencing and RNAasemismatch techniques (Winter et al., Proc. Natl. Acad. Sci. 82:7575-7579(1985)). Furthermore, one skilled in the art understands that, where thepresence or absence of multiple alleles or a fibrostenosis-predisposinghaplotype is to be determined, individual alleles can be detected by anycombination of molecular methods. See, in general, Birren et al. (Eds.)Genome Analysis: A Laboratory Manual Volume 1 (Analyzing DNA) New York,Cold Spring Harbor Laboratory Press (1997). In addition, one skilled inthe art understands that multiple alleles can be detected in individualreactions or in a single reaction (a “multiplex” assay). In view of theabove, one skilled in the art realizes that the methods of the inventionfor diagnosing or predicting susceptibility to a clinical subtype ofCrohn's disease characterized by fibrostenosing disease can be practicedusing one or any combination of the well known assays described above orknown in the art.

[0079] The present invention further provides a method of diagnosing orpredicting susceptibility to a clinical subtype of Crohn's diseasecharacterized by fibrostenosing disease by determining the presence orabsence in an individual of a fibrostenosis-predisposing haplotype,where the presence of the fibrostenosis-predisposing haplotype isdiagnostic of or predictive of susceptibility to the clinical subtype ofCrohn's disease characterized by fibrostenosing disease. In oneembodiment, the fibrostenosis-predisposing haplotype is associated witha clinical subtype of Crohn's disease characterized by fibrostenosingdisease with an odds ratio of at least 2 and a lower 95% confidencelimit greater than 1. In another embodiment, thefibrostenosis-predisposing haplotype is associated with a clinicalsubtype of Crohn's disease characterized by fibrostenosing disease withan odds ratio of at least 3, at least 4, at least 5, at least 6, and alower 95% confidence limit greater than 1.

[0080] The term “fibrostenosis-predisposing haplotype,” as used herein,means a combination of alleles that tend to be inherited together withthe clinical subtype of Crohn's disease characterized by fibrostenosingdisease more often than would be expected according to traditionalMendelian genetics. In a method of the invention, the clinical subtypeof Crohn's disease can be, for example, characterized by fibrostenosingdisease independent of small bowel involvement. In one embodiment, thefibrostenosis-predisposing haplotype includes at least one allele linkedto the NOD2/CARD15 locus. In another embodiment, thefibrostenosis-predisposing haplotype includes afibrostenosis-predisposing allele. In a further embodiment, afibrostenosis-predisposing haplotype contains, for example, a variantallele at the NOD2/CARD15 locus. In another embodiment, thefibrostenosis-predisposing haplotype includes the “2” allele at SNP 8,SNP 12, or SNP 13. In a futher embodiment, thefibrostenosis-predisposing haplotype includes the “2” allele at SNP 13.In still further embodiments, the fibrostenosis-predisposing haplotypeincludes the “2” allele at SNP 8, SNP 12, and SNP 13. In anotherembodiment, the fibrostenosis-predisposing haplotype includes a JW1,JW15, JW16, JW17, or JW18 variant allele. One skilled in the artunderstands that a fibrostenosis-predisposing haplotype can containalleles that individually are not significantly associated thefibrostenosing subtype of Crohn's disease, so long as the combination ofalleles making up the haplotype tend to be inherited together with thefibrostenosing subtype of Crohn's disease more often than would beexpected according to traditional Mendelian genetics.

[0081] The presence or absence of a fibrostenosis-predisposing haplotypecan be accomplished using any of the methods described herein above fordetermining the presence or absence of a fibrostenosis-predisposingallele. As an example, enzymatic amplification such as polymerase chainreaction amplification, for example, using one or more fluorescentlylabeled probes or one or more probes containing a DNA minor grove bindercan be useful for determining the presence or absence of afibrostenosis-predisposing haplotype in a method of the invention.

[0082] Antibody based methods also can be useful for determining thepresence or absence of a fibrostenosis-predisposing allele orfibrostenosis-predisposing haplotype in a method of the invention. As anexample, an antibody that is specifically reactive with a polypeptide orfragment thereof encoded by fibrostenosis-predisposing allele can beused to detect the presence or absence of that allele in an individual.Such an antibody can be, for example, specifically reactive with thetruncated version of NOD2/CARD15 generated by a “2” allele at SNP 13 butnot reactive with full-length or wild type NOD2/CARD15.

[0083] Antibodies useful in the methods of the invention include,without limitation, monoclonal and polyclonal antibodies, single chainantibodies, chimeric antibodies, bifunctional or bispecific antibodies,humanized antibodies, human antibodies, and complementary determiningregion (CDR)-grafted antibodies, including compounds which include CDRor antigen-binding sequences, which differentially bind to a polypeptideor fragment encoded by a fibrostenosis-predisposing allele but not toother, non-predisposing alleles. Antibody fragments, including Fab,Fab′, F(ab′)₂ and Fv, also can be useful in the methods of the inventionas can plastic antibodies or molecularly imprinted polymers (MIPs; Hauptand Mosbauch, TIBTech 16:468-475 (1998)). Screening assays to determinedifferential binding specificity of an antibody are well known in theart (see Harlow et al. (Eds), Antibodies: A Laboratory Manual; ColdSpring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988)).

[0084] Antibodies useful in a method of the invention can be producedusing any method well known in the art, using a polypeptide, orimmunogenic fragment thereof, encoded by a fibrostenosis-predisposingallele. Immunogenic polypeptides or fragments can be isolated fromnatural sources, from recombinant host cells, or can be chemicallysynthesized. Methods for synthesizing such peptides are known in the artas described, for example, in Merrifield, J. Amer. Chem. Soc. 85:2149-2154 (1963); Krstenansky et al., FEBS Lett. 211:10 (1987).

[0085] Antibodies that differentially bind to polypeptides encoded byfibrostenosis-predisposing alleles of the invention can be labeled witha detection moiety and used to detect the presence, absence or amount ofthe encoded polypeptide in vivo, in vitro, or in situ. A moiety, such asa fluorescent molecule, can be linked to an antibody for use in a methodof the invention. A moiety such as detection moiety can be linked to anantibody using, for example, carbodiimide conjugation (Bauminger andWilchek, Meth. Enzymol. 70:151-159 (1980)).

[0086] Antibodies that differentially bind to polypeptides encoded by afibrostenosis-predisposing allele can be used in immunoassays.Immunoassays include, without limitation, radioimmunoassays,enzyme-linked immunosorbent assays (ELISAs) and immunoassays withfluorescently labeled antibodies, which are well known in the art.Antibodies can also be used to detect the presence or absence of apolypeptide of interest in a cell or tissue using immunohistochemistryor other in situ assays. Furthermore, cells containing a polypeptide ofinterest either on the surface of the cell or internally can be detectedby an antibody using assays such as fluorescence activated cell sorting(FACS). One skilled in the art understands that these and other routineassays can be useful for determining the presence or absence of the geneproduct of a fibrostenosis-predisposing allele according to a method ofthe invention.

[0087] The methods of the invention optionally include generating areport indicating the presence or absence in a individual of afibrostenosis-predisposing allele or fibrostenosis-predisposinghaplotype. The methods of the invention also optionally includegenerating a report indicating the presence or absence in the individualof a clinical subtype of Crohn's disease characterized by fibrostenosingdisease or the risk that an individual has of having or developing thefibrostenosing subtype of Crohn's disease.

[0088] A report can be in a variety of forms, for example, a report canbe printed on paper or a report can be an electronic report that is notprinted but is transmitted over an electronic medium such as electronicmail or a computer diskette. A report also can be an oral report thatindicates the presence or absence in the individual of afibrostenosis-predisposing allele or a clinical subtype of Crohn'sdisease characterized by fibrostenosing disease.

[0089] The invention also provides a method of optimizing therapy in anindividual by determining the presence or absence in the individual of afibrostenosis-predisposing allele linked to a NOD2/CARD15 locus,diagnosing individuals in which the fibrostenosis-predisposing allele ispresent as having a fibrostenosing subtype of Crohn's disease, andtreating the individual having a fibrostenosing subtype of Crohn'sdisease based on the diagnosis. Treatment for the fibrostenosing subtypeof Crohn's disease currently includes, for example, surgical removal ofthe affected, strictured part of the bowel. In one embodiment, thepresence or absence of a fibrostenosis-predisposing allele is determinedin an individual having a known diagnosis of Crohn's disease. In anotherembodiment, the diagnosis is recorded in the form of a report asdescribed above.

[0090] The following examples are intended to illustrate but not limitthe present invention.

EXAMPLE I Selection and Characterization of Study Subjects

[0091] This example describes the clinical characterization of how twocohorts of Crohn's disease patients.

[0092] A. Selection of Study Subjects

[0093] Two cohorts of Crohn's disease patients were consecutivelyidentified from an inflammatory bowel disease referral center(Cedars-Sinai Medical Center Inflammatory Bowel Disease Center). Thefirst cohort of 142 patients, ascertained between 1993-1996 anddesignated “CD1”, was previously described in Vasiliauskas et al., Gut47:487-496 (2000)). The second cohort of 59 patients (Cohort 2) wascollected between 1999-2001 and designated “CD2”. A cohort of 175patients with ulcerative colitis was used as an inflammatory boweldisease control group. This study, which was reviewed and approved forhuman subject participation by the Cedars-Sinai Institutional ReviewBoard, involved the collection of clinical, serologic and genetic datafrom patients consenting to the study.

[0094] A diagnosis of Crohn's disease in the cohort patients was definedby the presence of a combination of established features from at leasttwo of the following categories: 1) clinical—perforating or fistulizingdisease, obstructive symptoms secondary to small bowel stenosis orstricture; 2) endoscopic—deep linear or serpiginous ulcerations,discrete ulcers in normal-appearing mucosa, cobblestoning, ordiscontinuous or asymmetric inflammation; 3) radiographic—segmentaldisease (skip lesions), small bowel or colon strictures, stenosis, orfistula, and; 4) histopathologic—submucosal or transmural inflammation,multiple granulomas, marked focal cryptitis or focal chronicinflammatory infiltration within and between biopsies, or skip lesionsincluding rectal sparing in the absence of local therapy.

[0095] To identify clinical features and immunological traits associatedwith allelic variants of the NOD2/CARD15 gene, the study was designed toanalyze two consecutively ascertained cohorts of patients with Crohn'sdisease. The first cohort was used to explore the relationship ofNOD2/CARD15 alleles with an array of clinical and serologic variables,thereby generating hypotheses. The second cohort was then used toconfirm the specific hypotheses generated from analysis of the firstcohort. To minimize type I error and maximize statistical power, thesignificance of the associations in the first cohort were permitted tobe less stringent (p<0.1) than in the second cohort (p<0.05). Byavoiding a highly stringent correction for the number of variablesexamined in the first cohort, there was an increased ability to identifyspecific associations between NOD2/CARD15 allele variants and clinicalvariables.

[0096] B. Clinical Characterization of Study Subjects

[0097] Patients with Crohn's disease were characterized as havingfibrostenosing disease, internal-perforating disease, perianalfistulizing disease or ulcerative colitis (UC)-like disease based onpreviously described criteria (Gasche et al., Inflammatory BowelDiseases 6:8-15 (2000); Vasiliauskas et al., Gut 47:487-496 (2000);Vasiliauskas et al., Gastroenterology 110:1810-1819 (1996); andGreenstein et al., Gut 29:588-592 (1988)). According to well establishedcriteria, fibrostenosing disease was defined by documented persistentintestinal obstruction or an intestinal resection for an intestinalobstruction. Internal perforating disease was defined as current orprevious evidence of entero-enteric or entero-vesicular fistulae,intraabdominal abscesses, or small bowel perforation. Perianalperforating disease was defined by current or previous evidence ofeither perianal fistulae or abscesses or rectovaginal fistula. UC-likedisease was defined by current or previous evidence of left-sidedcolonic involvement, symptoms of bleeding or urgency, and cryptabscesses on colonic biopsies as previously described. Disease locationwas classified as small bowel, colon, or both based on one or moreendoscopic, radiologic or pathologic studies. A panel of inflammatorybowel disease physicians unaware of the results of serologic and genetictesting reached a consensus on phenotype based on the clinical data.

[0098] Serum ANCA expression and ANCA subtype characterization wereperformed by fixed neutrophil enzyme-linked immunosorbent assay (ELISA)as previously described (Saxon et al., J. Allergy Clin. Immunol.86:202-210 (1990)). Briefly, human peripheral blood neutrophils fixedwith methanol were reacted with control and coded sera at a 1:100dilution. Anti-human immunoglobulin G (IgG) (chain-specific) antibody(Jackson Immunoresearch Labs, Inc.; West Grove, Pa.) conjugated toalkaline phosphatase was added to label neutrophil-bound antibody, and acalorimetric reaction was performed. Levels were determined relative toa standard consisting of pooled sera obtained from well-characterizedpANCA+ UC patients. Results were expressed as ELISA units (EU) permilliliter. ANCA+ sera were further subtyped via indirectimmunofluorescent staining to determine the ANCA neutrophil bindingpattern, as previously described (Saxon et al., supra, 1990). Serashowing the characteristic perinuclear highlighting and losing thecharacteristic staining pattern when treated with deoxyribonuclease weretermed pANCA+ (Vidrich et al., J. Clin. Immunol. 15:293-299 (1995)). Forthe purposes of this study, patients were considered pANCA+ if they wereboth positive for ANCA by ELISA and lost perinuclear immunofluorescencestaining with deoxyribonuclease treatment.

[0099] Sera were analyzed for ASCA expression in a blinded fashion byusing a fixed ELISA assay (Vasiliauskas et al., Gut 47:487-496 (2000);and Vermeire et al., Gastroenteroloqy 120:827-833 (2001)). Two patientsin the second cohort did not undergo ASCA testing. High-bindingpolystyrene microtiter plates were coated with purifiedphosphopeptidomannans extracted from yeast S. uvarum, a subspecies of S.cerevisiae. Coded patient sera were diluted and added to the wells,followed by an enzyme-linked calorimetric reaction. Color developmentwas proportional to concentrations of ASCA antibody present in the sera.Levels were determined and results expressed as EU per milliliter,relative to a standard, which was derived from a pool of patient serawith well-characterized Crohn's disease found to have reactivity to thisantigen. Sera showing ASCA IgG reactivity of >40 EU/mL or IgA reactivityof >20 EU/mL were termed ASCA+. Serological assays were performed atCedars-Sinai Medical Center and Prometheus Laboratories (San Diego,Calif.) using substantially identical methodology. The clinicalcharacteristics of the two Crohn's disease cohorts are shown in Table 1.TABLE 1 Clinical Characteristics of Two Crohn's Disease Cohorts ClinicalCD1 CD2 characteristics n = 142 n = 59 p Gender (M/F) 79/63 33/26 0.969Age at onset 22 (4-67) 22(2-58) 0.6621 Ethnicity 60/82 23/36 0.668(Jew/Non-Jew) Disease location (%) SB only 19.0 26.4 0.496 Colon only20.4 20.8 SB and Colon 60.6 52.8 Perianal perforating 35.9 28.8 0.332(%) Internal perforating 47.2 23.7 0.002 (%) Fibrostenosing 59.9 30.50.001 disease (%) UC-like (%) 39.4 22.0 0.018 pANCA-positive (%) 19.712.5 0.295 ASCA-positive* (%) 57.0 38.6 0.019

EXAMPLE II Patients with Crohn's Disease Have an Increased Frequency ofRare Allelic Variants of NOD2/CARD15

[0100] This example describes the association of a “2” allele at SNP 8,SNP 12, or SNP 13 within the NOD2/CARD15 locus (rare allelic variants ofNOD2/CARD15) with Crohn's disease in a North American population.

[0101] In order to determine whether the North American Crohn's diseasepatient populations in Cohorts 1 and 2 expressed allelic variants ofNOD2/CARD15, Cohort 1 (hypothesis-generating) and Cohort 2(hypothesis-confirming) were genotyped for the rare allelic variant ofSNP 8 (R675W), SNP 12 (G881R) and SNP 13 (3020insC). A cohort ofulcerative colitis patients was used for comparison.

[0102] Genotyping was performed using a genotyping assay employing5′-exonuclease technology, the TaqMan MGB™ assay (PE Biosystems; FosterCity, Calif.). Primers were designed using the software PrimerExpress1.5™ (PE Biosystems) and sequence information found in dbSNP forNOD2/CARD15 SNP 5, 8, 12, and 13. The MGB™ design adds a “minor groovebinder” to the 3′ end of the TaqMan™ probes, thereby increasing thebinding temperature of the probe and enabling the use of shorter probesthan in conventional Taqman™ assays (Kutyavin et al., Nucleic Acids Res.25:3718-3723 (1997)). This has the effect of increasing thediscrimination between the alleles in the assay (Kutyavin et al.,Nucleic Acids Res. 28:655-661 (2000)). Assays were performed followingthe manufacturer's recommendations (PE Biosystems bulletin 4317594) inan ABI 7900 instrument. Genotyping was performed blinded to clinicalstatus of the subjects. Primers and probes used in the genotyping assayare shown in Tables 2 and 3. TABLE 2 Primers Used in Taqman MGB™ Assayfor SNPs 5, 8, 12 and 13 SNP SEQ ID Primer Forward Primer Reverse PrimerNO 5 5′ GGTGGCTGGGCT 5′ CTCGCTTCCTCAGTACCT for 37 CTTCT 3′ ATGATG 3′ rev38 8 5′ CTGGCTGAGTGC 5′ GGCGGGATGGAGTGGAA for 39 CAGACATCT 3′ 3′ rev 4012 5′ CCACCTCAAGCT 5′ GTTGACTCTTTTGGCCTT for 41 CTGGTGATC 3′ TTCAG 3′rev 42 13 5′ CCTTACCAGACT 5′ TGTCCAATAACTGCATCA for 43 TCCAGGATGGT 3′CCTACCT 3′ rev 44

[0103] TABLE 3 TAQMAN PROBES Allele Seq ID detected Probe sequence NOSNP5 6FAM-CATGGCTGGACCC-MGBNFQ 45 wild type allele (“1”) SNP5TET-CATGGCTGGATCC-MGBNFQ 46 variant allele (“2”) SNP86FAM-TGCTCCGGCGCCA-MGBNFQ 47 wild type allele (“1”) SNP8TET-CTGCTCTGGCGCCA-MGBNFQ 48 variant allele (“2”) SNP126FAM-CTCTGTTGCCCCAGAA-MGBNFQ 49 wild type allele (“1”) SNP 12TET-CTCTGTTGCGCCAGA-MGBNFQ 50 wild type allele (“1”) SNP 13TET-CTTTCAAGGGCCTGC-MGBNFQ 51 wild type allele (“1”) SNP136FAM-CCTTTCAAGGGGCCT-MGBNFQ 52 variant allele (“2”) JW16FAM-AAGACTCGAGTGTCCT-MGBNFQ 53 wild type allele JW1VIC-AGACTCAAGTGTCCTC-MGBNFQ 54 variant

[0104] As shown in Table 4, each of three rare allelic variants ofNOD2/CARD15 (a “2” allele at SNP 8, SNP 12, or SNP 13) was significantlymore frequent in patients with Crohn's disease compared with ulcerativecolitis. In addition, as can be seen in Table 4, the frequency of eachof the NOD2/CARD15 rare allelic variants was similar in each cohort ofCrohn's disease patients, supporting the combined use of the twocohorts. The overall frequency of any of the three NOD2/CARD15 rareallelic variants was 35% in Crohn's disease patients compared with 11%in ulcerative colitis patients (p=0.001).

[0105] Within the combined Crohn's disease cohort, the frequency ofhomozygotes with a “2” allele at SNP 13 (3020insC) and compound rareallelic heterozygotes was 1% and 4%, respectively, while none of theulcerative colitis patients had such a genotype. These resultsdemonstrate that rare allelic variants of NOD2/CARD15 are associatedwith Crohn's disease across diverse geographic and ethnically-definedpatient populations. TABLE 4 Frequency of NOD2/CARD15 Rare AllelicVariants in CD and UC Patient Populations Combined p CD1 and (UC vs.Allelic UC CD1 CD2 CD2 Combined variants (n = 175) (n = 142) (n = 59) (n= 201) CD) R675W 5.7% 16.9% 15.3% 16.4% 0.001 (SNP 8 “2” allele) G881R1.7% 12.0% 10.2% 11.4% 0.0001 (SNP 12 “2” allele) 3020insC 3.4% 11.3%11.9% 11.4% 0.004 (SNP 13 “2” allele) Carriage of 10.9%  36.6% 32.2%35.3% 0.001 any allelic variant

EXAMPLE III Rare Variant Alleles in the NOD2/CARD15 Locus are Associatedwith the Fibrostenosing Subtype of Crohn's Disease in Cohort 1

[0106] This example demonstrates that a “2” allele at SNP 8, SNP 12, orSNP 13 is significantly associated with fibrostenosing disease in Cohort1.

[0107] Patients with Crohn's disease express diverse clinical phenotypesthat can be due to differences in underlying genetic factors. In orderto determine whether rare variant alleles at the NOD2/CARD15 locus wereassociated with specific Crohn's disease-related clinical phenotypes ordisease-related serum immune markers, univariate analysis was performed.The univariate analysis evaluated the association between NOD2/CARD15allelic variants at SNP 8, SNP 12, or SNP 13 and predefined clinicalcharacteristics, including age of onset, disease location, and diseasephenotype (fibrostenosing disease, internal-perforating disease,perianal fistulizing disease or ulcerative colitis-like disease). Theassociation between NOD2/CARD15 allelic variants and expression of theserum immune markers ASCA and pANCA was also tested.

[0108] As shown in Table 5, univariate analysis indicated that a “2”allele at SNP 8, SNP 12, or SNP 13 of the NOD2/CARD15 locus wassignificantly associated with fibrostenosing disease in Cohort 1(p=0.049) for the three allelic variants combined. A positiveassociation at a less stringent significance level (p<0.1) was alsoobserved with small bowel involvement and younger age of onset, and anegative association was observed with UC-like disease in this cohort.With respect to serologic markers, patients with the “2” allele at SNP13 were more likely to express ASCA (p=0.053). These results demonstratethat a “2” allele at SNP 8, SNP 12, or SNP 13 is significantlyassociated with fibrostenosing disease in Cohort 1.

[0109] Statistical analysis was performed using SAS computer software(Version 6.10; SAS Institute, Inc.; Cary, N.C.). Quantitative variableswere described as medians with a range. Nonparametric statistical testswere used to test differences in quantitative variables between twogroups. A Chi-square test or Fisher's exact test (when the expectednumber was less than 5) was used to evaluate associations betweencarriers and non-carriers of the rare alleles or between genotypes andcategorical variables, such as type of IBD, disease location, diseasebehavior, and antibody positivity. In addition, a Mantel Haenszelstratified association test was performed for all genotype and phenotypeassociations by controlling for potential confounding effect due toethnic variation. This stratified association test was also used toevaluate whether the small bowel involvement and fibrostenosing diseasewere independently associated with NOD2/CARD15 variants (see Example IXbelow). TABLE 5 Relationship of NOD2/CARD15 Rare Variant Alleles andClinical Phenotypes of Crohn's Disease in Cohort 1 Qualitative Trait %NOD2 variant carriers Carriage of Clinical R675W G881R 3020insC anyallelic phenotypes n (SNP 8) (SNP 12) (SNP 13) variant Small bowel yes113 19.47% 11.50% 14.16% 40.71% involvement no 29  6.90% 13.79%  0.00%20.69% p value  0.081  0.494  0.04  0.063 Perianal yes 51 11.76% 11.76%13.73% 35.29% perforating no 91 19.78% 12.09%  9.89% 37.36% p value 0.248  0.839  0.547  0.747 Internal yes 67 13.43% 16.42% 11.94% 37.31%perforating no 75 20.00%  8.00% 10.67% 36.00% p value  0.346  0.178 0.91  0.96 Fibrostenosing yes 85 18.82% 14.12% 15.29% 43.53% no 5714.04%  8.77%  5.26% 26.32% p value  0.389  0.458.  0.084  0.049 UC-likeyes 56 17.86% 10.71%  5.36% 30.36% no 86 16.28% 12.79% 15.12% 40.70% pvalue  0.822  0.736  0.076  0.22 pANCA yes 28 17.86% 14.29%  7.14%32.14% positive no 114 16.67% 11.40% 12.28% 37.72% p value  0.82  0.793 0.394  0.529 ASCA yes 81 18.52%  9.88% 16.05% 38.27% positive no 6114.75% 14.75%  4.92% 34.43% p value  0.467  0.234  0.053  0.744Quantitative Trait Carriage median (range) of R675W G881R 3020insC anyallelic (SNP 8) (SNP 12) (SNP 13) variant Age of onset yes 22 22 19 20carrier of (6-67) (4-62) (10-50) (4-67) NOD2 variant no 22 22 22 22(4-63) (4-67) (4-67) (4-63) p  0.715  0.937  0.074  0.238

EXAMPLE IV Rare Variant Alleles in the NOD2/CARD15 Locus are Associatedwith the Fibrostenosing Subtype of Crohn's Disease in Cohort 2

[0110] This example demonstrates that a “2” allele at SNP 8, SNP 12, orSNP 13 of the NOD2/CARD15 locus is significantly associated withfibrostenosing disease in Cohort 2.

[0111] The results obtained with Cohort 1 in Example III indicated thatNOD2/CARD15 rare variant alleles were positively associated withfibrostenosing Crohn's disease, small bowel involvement, ASCApositivity, and younger age of onset, and negatively associated withUC-like disease (see Table 5). These hypotheses were further testedusing Cohort 2; the results are shown in Table 6. As with Cohort 1,Cohort 2 demonstrated a significant association between a “2” allele atSNP 8, SNP 12, or SNP 13 of the NOD2/CARD15 locus and fibrostenosingdisease (p=0.002, with Bonferroni correction p=0.01; see Table 6). Theseresults indicate that a “2” allele at SNP 8, SNP 12, or SNP 13 of theNOD2/CARD15 locus is significantly associated with fibrostenosingdisease in Cohort 2. TABLE 6 Relationship of NOD2/CARD15 Rare VariantAlleles and Clinical Phenotypes of Crohn's Disease in Cohort 2Qualitative Trait % NOD2 variant carriers Clinical R675W G881R 3020insCCarriage of any phenotypes n (SNP 8) (SNP 12) (SNP 13) allelic variantFibrostenos- yes 18 22.22% 22.22% 27.78% 61.11% ing no 41 12.20%  4.88% 4.88% 19.51% p  0.315  0.048  0.018  0.002 Small bowel yes 42 19.05% 9.52% 14.29% 35.71% involvement no 17  5.88% 11.76%  5.88% 23.53% p 0.22  0.828  0.288  0.354 ASCA yes 22  9.09% 13.64% 13.64% 31.82%positive no 35 17.14%  8.57% 11.43% 31.43% p  0.4  0.542  0.735  0.956Quantitative Trait median (range) R675W G881R 3020ins C Carriage of any(SNP 8) (SNP 12) (SNP 13) allelic variant Age of onset yes 27 26 17 22carrier of (10-58) (7-33) (13-35) (7-58) NOD2 no 19 20 24 22 variant(2-55) (2-58) (2-58) (2-55) p  0.332  0.9  0.566  0.981

EXAMPLE V Rare Variant Alleles in the NOD2/CARD15 Locus are Associatedwith the Fibrostenosing Subtype of Crohn's Disease in a Combined CohortRepresenting Cohorts 1 and 2

[0112] This example demonstrates that a “2” allele at SNP 8, SNP 12, orSNP 13 is significantly associated with fibrostenosing disease in acombined cohort representing cohorts 1 and 2.

[0113] As seen in FIG. 2, the association between a “2” allele at SNP 8,SNP 12, or SNP 13 of the NOD2/CARD15 locus (NOD2 variant carrier) andfibrostenosing disease was even more significant (p=0.001) when the twocohorts were analyzed together than when analyzed separately.

[0114] The association between a “2” allele at SNP 8, SNP 12, or SNP 13of the NOD2/CARD15 locus (NOD2 variant carrier) and fibrostenosingdisease was observed in both Jewish and non-Jewish individuals.Approximately 46% of Crohn's disease patients with fibrostenosingdisease (Jews 52% vs. non-Jews 42%) had at least one of these rarealleles compared with only 23% (Jews 21.6% vs. non-Jews 25%) of Crohn'sdisease patients without fibrostenosing disease (Odds ratio, 2.8; 95%Confidence interval, 1.56-5.18). Of the three rare variant alleles, theframeshift mutation 3020insC (a “2” allele at SNP 13) demonstrated thegreatest association with fibrostenosing disease (47% vs. 17%, p=0.006for cohorts combined). These results indicate that rare variant allelesin the NOD2/CARD15 locus are associated with the fibrostenosing subtypeof Crohn's disease in a combined cohort representing Cohorts 1 and 2.

EXAMPLE VI Crohn's Disease Patients with Homozygous Mutations orCompound Heterozygous Mutations in NOD2/CARD15

[0115] This example describes the increased risk of fibrostenosingdisease in Crohn's disease patients carrying homozygous mutations orcompound heterozygous mutations in NOD2/CARD15 locus.

[0116] As shown in FIG. 3, compared with patients who were not carriersof NOD2/CARD15 mutations at SNP 8, SNP 12 or SNP 13, patients who werecarriers of two mutations in NOD2/CARD15 were significantly more likelyto have fibrostenosing disease (85% vs. 43%; odds ratio 7.4; 95%confidence interval 1.9-28.9, p=0.004). Patients who were carriers of asingle NOD2/CARD15 mutation were also significantly more likely to havefibrostenosing disease when compared with patients who were not carriersof these NOD2/CARD15 mutations (64% vs. 43%; Odds ratio 2.37; 95%Confidence interval 1.26-4.47; p=0.008). These results indicate thatCrohn's disease patients with homozygous mutations or compoundheterozygous mutations in NOD2/CARD15 have an increased risk offibrostenosing disease.

EXAMPLE VII Fibrostenosing Disease Only Compared to Fibrostenosing andPerforating Disease

[0117] This example describes the association of the “2” allele at SNP8, SNP 12, or SNP 13 of the NOD2/CARD15 locus in patients withfibrostenosing disease compared to patients with fibrostenosing andperforating disease.

[0118] Fibrostenosing and perforating disease often occur in the samepatient. Patients with fibrostenosing disease can be characterized as i)having only fibrostenosing disease or ii) having both fibrostenosing andperforating disease. In order to address these phenotypes individually,patients in each of the two cohorts were separated by the presence offibrostenosing disease with perforating complications (Fib+perf) orwithout perforating complications (Fib only) and compared with patientswith perforating complications without evidence of fibrostenosis (Perfonly). The percentage of patients having only fibrostenosing diseasethat carried a “2” allele at SNP 8, SNP 12, or SNP 13 of the NOD2/CARD15locus was 48.3%, which was similar to that seen in patients with bothfibrostenosing and perforating complications (46.0%; p=0.8). As seen inFIG. 3, when patients with fibrostenosing disease were compared withthose patients described as having perforating disease only (perianal orinternal), the frequency of the “2” allele at SNP 8, SNP 12 or SNP 13 ofthe NOD2/CARD15 locus in patients with fibrostenosing disease (with orwithout perforating complications) was significantly greater than thatseen in patients with only perforating complications (46.6% versus18.6%; p=0.002).

EXAMPLE VIII Multivariant Analysis of the Combined Patient Cohort

[0119] This example demonstrates that fibrostenosing disease isindependently associated with a “2” allele at SNP 8, SNP 12, or SNP 13of the NOD2/CARD15 locus.

[0120] For multivariant analysis, all variables with at least borderlinesignificance (p<0.1) in either cohort were tested simultaneously fortheir association with a “2” allele at SNP 8, SNP 12, or SNP 13 of theNOD2/CARD15 locus by using logistic regression. As shown in Table 7, theclinical phenotype of fibrostenosing disease was significantlyassociated (p<0.05) with these rare alleles at NOD2/CARD15 locus (OR2.8; 95% CI, 1.3-6.0). These results confirm that fibrostenosing diseaseis independently associated with a “2” allele at SNP 8, SNP 12, or SNP13 of the NOD2/CARD15 locus. TABLE 7 Multivariate Analysis in theCombined Cohort for 5 Phenotypic Variables Clinical phenotypes OR 95% CIP Fibrostenosing disease 2.8 1.3-6.0 0.011 Small bowel involvement 1.30.5-3.4 0.561 UC-like 0.9 0.4-1.7 0.658 ASCA positive 0.7 0.3-1.3 0.250Age of onset 1.0 0.9-1.0 0.874

EXAMPLE IX Fibrostenosing Disease and Small-Bowel Involvement

[0121] This example demonstrates that the association betweenfibrostenosing disease and a “2” allele at SNP 8, SNP 12, or SNP 13 ofthe NOD2/CARD15 locus is independent of small-bowel involvement.

[0122] Because fibrostenosing disease is more likely to occur inpatients with small-bowel involvement, patients were stratified on thebasis of small-bowel involvement to analyze whether the associationbetween fibrostenosing disease and NOD2/CARD15 variant alleles was aprimary association. Among patients with small-bowel involvement, 26.4%of patients who did not have fibrostenosing disease (n=53) had a “2”allele at SNP 8, SNP 12, or SNP 13, whereas 46.1% of patients who hadfibrostenosing disease (n=102) had a “2” allele at SNP 8, SNP 12, or SNP13 (p=0.017). A similar trend was observed among patients withoutsmall-bowel involvement (p=0.05), and the combined analysis conditioningon small-bowel involvement yielded a significance level of 0.009.

[0123] After controlling for fibrostenosing disease, small-bowelinvolvement was not associated with a “2” allele at SNP 8, SNP 12, orSNP 13 of the NOD2/CARD15 locus (p=0.63). This result agrees with theresults from logistic regression analysis (see Example VIII) andindicates that the association between fibrostenosing disease and a “2”allele at SNP 8, SNP 12, or SNP 13 of the NOD2/CARD15 locus isindependent of small-bowel involvement. These results further indicatethat the observed small-bowel association with a “2” allele at SNP 8,SNP 12, or SNP 13 of the NOD2/CARD15 locus is secondary to the presenceof fibrostenosing disease.

[0124] All journal article, reference, and patent citations providedabove, in parentheses or otherwise, whether previously stated or not,are incorporated herein by reference.

[0125] Although the invention has been described with reference to theexamples above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1 67 1 494 DNA Homo sapiens 1 accttcagat cacagcagcc ttcctggcagggctgttgtc ccgggagcac tggggcctgc 60 tggctgagtg ccagacatct gagaaggccctgctccggcg ccaggcctgt gcccgctggt 120 gtctggcccg cagcctccgc aagcacttccactccatccc gccagctgca ccgggtgagg 180 ccaagagcgt gcatgccatg cccgggttcatctggctcat ccggagcctg tacgagatgc 240 aggaggagcg gctggctcgg aaggctgcacgtggcctgaa tgttgggcac ctcaagttga 300 cattttgcag tgtgggcccc actgagtgtgctgccctggc ctttgtgctg cagcacctcc 360 ggcggcccgt ggccctgcag ctggactacaactctgtggg tgacattggc ctggagcagc 420 tgctgccttg ccttggtgtc tgcaaggctctgtagtgagt gttactgggc attgctgttc 480 aggtatgggg gagc 494 2 494 DNA Homosapiens 2 gctcccccat acctgaacag caatgcccag taacactcac tacagagccttgcagacacc 60 aaggcaaggc agcagctgct ccaggccaat gtcacccaca gagttgtagtccagctgcag 120 ggccacgggc cgccggaggt gctgcagcac aaaggccagg gcagcacactcagtggggcc 180 cacactgcaa aatgtcaact tgaggtgccc aacattcagg ccacgtgcagccttccgagc 240 cagccgctcc tcctgcatct cgtacaggct ccggatgagc cagatgaacccgggcatggc 300 atgcacgctc ttggcctcac ccggtgcagc tggcgggatg gagtggaagtgcttgcggag 360 gctgcgggcc agacaccagc gggcacaggc ctggcgccgg agcagggccttctcagatgt 420 ctggcactca gccagcaggc cccagtgctc ccgggacaac agccctgccaggaaggctgc 480 tgtgatctga aggt 494 3 540 DNA Homo sapiens 3 atcaaaaccctgagaggaca agggacattt ccaagtcacc cagaaagact cgagtgtcct 60 ctcttgaaatccaatggtct tttttcctta ctccattgcc taacattgtg gggtagaaat 120 aaagttcaaagaccttcaga actggcccca gctcctccct cttcacctga tctccccaag 180 aaaactgcaggatagactct gaagcttacc tgagccacct caagctctgg tgatcaccca 240 aggcttcagccagggcctgg gccccctcgt cacccactct gttgccccag aatctgaaaa 300 ggccaaaagagtcaacagac agtgtcagtg agtacctgat atgtgttcta gacatgaact 360 aacagtcctcctccctctgc agtcccagcc agaggggcag gaccactcaa tcccagagtg 420 gcctcactggggctcctggt cccagcaaag tggacctgcc tccatctttt gggtgggatg 480 gccaaacttaacccaagagt tttcagtggc tttacattac agacttagag aatagtagag 540 4 540 DNAHomo sapiens 4 ctctactatt ctctaagtct gtaatgtaaa gccactgaaa actcttgggttaagtttggc 60 catcccaccc aaaagatgga ggcaggtcca ctttgctggg accaggagccccagtgaggc 120 cactctggga ttgagtggtc ctgcccctct ggctgggact gcagagggaggaggactgtt 180 agttcatgtc tagaacacat atcaggtact cactgacact gtctgttgactcttttggcc 240 ttttcagatt ctggggcaac agagtgggtg acgagggggc ccaggccctggctgaagcct 300 tgggtgatca ccagagcttg aggtggctca ggtaagcttc agagtctatcctgcagtttt 360 cttggggaga tcaggtgaag agggaggagc tggggccagt tctgaaggtctttgaacttt 420 atttctaccc cacaatgtta ggcaatggag taaggaaaaa agaccattggatttcaagag 480 aggacactcg agtctttctg ggtgacttgg aaatgtccct tgtcctctcagggttttgat 540 5 541 DNA Homo sapiens 5 tttaaaaatg aaatcattgc tccctacttaaagaggtaaa gacttctttc ttagacagag 60 aatcagatcc ttcacatgca gaatcattctcactgaatgt cagaatcaga agggatcctc 120 aaaattctgc cattcctctc tcccgtcaccccattttaca gatagaaaaa ctgaggttcg 180 gagagctaaa acaggcctgc ccaggggccttaccagactt ccaggatggt gtcattcctt 240 tcaaggggcc tgcaggaggg cttctgcccctaggtaggtg atgcagttat tggacaacct 300 ggaaaagaag atacaatggt gagcttcaaggattcttggt tttcctcttg aaactgtcca 360 gttaaagaga ctgcaggagt tagccagtctactgaagccc acctgtccct tagacacatc 420 ctgctcatgt ctgagattcc caatgagctcatcaacaaag gctcagtacc atcagtgaaa 480 tgtaaccgtc tctcttccat tcactagatgagtttatcaa attaagtagc cactccctta 540 g 541 6 541 DNA Homo sapiens 6ctaagggagt ggctacttaa tttgataaac tcatctagtg aatggaagag agacggttac 60atttcactga tggtactgag cctttgttga tgagctcatt gggaatctca gacatgagca 120ggatgtgtct aagggacagg tgggcttcag tagactggct aactcctgca gtctctttaa 180ctggacagtt tcaagaggaa aaccaagaat ccttgaagct caccattgta tcttcttttc 240caggttgtcc aataactgca tcacctacct aggggcagaa gccctcctgc aggccccttg 300aaaggaatga caccatcctg gaagtctggt aaggcccctg ggcaggcctg ttttagctct 360ccgaacctca gtttttctat ctgtaaaatg gggtgacggg agagaggaat ggcagaattt 420tgaggatccc ttctgattct gacattcagt gagaatgatt ctgcatgtga aggatctgat 480tctctgtcta agaaagaagt ctttacctct ttaagtaggg agcaatgatt tcatttttaa 540 a541 7 540 DNA Homo sapiens 7 aacagcagtg ctcaaagagt agagtccgca cagagagtggtttggccatg cactgcagct 60 gccggcagct gaatgggaag acaaagagaa attcctggaagtcttgccct gcagcccaca 120 gcaagtgcag ccgctgcagg agcgtgctct tgccactgcccgcctcaccc accaccagca 180 cagtgtccgc atcgtcattg aggtggccag gggtgctgaagagctcctcc aggcccaggg 240 tggctgggct cttctgcggg ggtccagcca tgcccacatctgcccagacc tccaggacat 300 tctctgtgta tatgtcctcc aggcagagcg tctctgctccatcataggta ctgaggaagc 360 gagactgagc agacaccgtg gtcctcagct tggccatatacttcttgcat gtggcagctg 420 gaaggcagaa gaagaggcag atgaaggtgg caccatggtgaagacgggac ctaaccagac 480 aatgggctgc tgcgggggac gctgacataa ctgaagggataggagagcca gcgggcgccc 540 8 540 DNA Homo sapiens 8 gggcgcccgc tggctctcctatcccttcag ttatgtcagc gtcccccgca gcagcccatt 60 gtctggttag gtcccgtcttcaccatggtg ccaccttcat ctgcctcttc ttctgccttc 120 cagctgccac atgcaagaagtatatggcca agctgaggac cacggtgtct gctcagtctc 180 gcttcctcag tacctatgatggagcagaga cgctctgcct ggaggacata tacacagaga 240 atgtcctgga ggtctgggcagatgtgggca tggctggacc cccgcagaag agcccagcca 300 ccctgggcct ggaggagctcttcagcaccc ctggccacct caatgacgat gcggacactg 360 tgctggtggt gggtgaggcgggcagtggca agagcacgct cctgcagcgg ctgcacttgc 420 tgtgggctgc agggcaagacttccaggaat ttctctttgt cttcccattc agctgccggc 480 agctgcagtg catggccaaaccactctctg tgcggactct actctttgag cactgctgtt 540 9 520 DNA Homo sapeins 9gcactgggca cccactacca atggattgga attggtcctt aagataaaat gtacctgatc 60cagcccaata tcttcaattt acagatactg tatcaaaacc ctgagaggac aagggacatt 120tccaagtcac ccagaaagac tcgagtgtcc tctcttgaaa tccaatggtc ttttttcctt 180actccattgc ctaacattgt ggggtagaaa taaagttcaa agaccttcag aactggcccc 240agctcctccc tcttcacctg atctccccaa gaaaactgca ggatagactc tgaagcttac 300ctgagccacc tcaagctctg gtgatcaccc aaggcttcag ccagggcctg ggccccctcg 360tcacccactc tgttgcccca gaatctgaaa aggccaaaag agtcaacaga cagtgtcagt 420gagtacctga tatgtgttct agacatgaac taacagtcct cctccctctg cagtcccagc 480cagaggggca ggaccactca atcccagagt ggcctcactg 520 10 520 DNA Homo sapiens10 cagtgaggcc actctgggat tgagtggtcc tgcccctctg gctgggactg cagagggagg 60aggactgtta gttcatgtct agaacacata tcaggtactc actgacactg tctgttgact 120cttttggcct tttcagattc tggggcaaca gagtgggtga cgagggggcc caggccctgg 180ctgaagcctt gggtgatcac cagagcttga ggtggctcag gtaagcttca gagtctatcc 240tgcagttttc ttggggagat caggtgaaga gggaggagct ggggccagtt ctgaaggtct 300ttgaacttta tttctacccc acaatgttag gcaatggagt aaggaaaaaa gaccattgga 360tttcaagaga ggacactcga gtctttctgg gtgacttgga aatgtccctt gtcctctcag 420ggttttgata cagtatctgt aaattgaaga tattgggctg gatcaggtac attttatctt 480aaggaccaat tccaatccat tggtagtggg tgcccagtgc 520 11 535 DNA Homo sapiens11 tcactaacca gctcaggaag ctcaccagct tgggaagtta atcattatgt ctagcttcag 60tttctcctgc ttcagtttaa attgggaaag agagagaaaa aatattcact cattatctgt 120ttcctaaaat tgtccttaac atccttcctc ttactccttt attacctggt cgggcttccc 180ctcttcaggc gaaatctgtc agtctatctg cattgccttt tgatctctac ttcagttact 240acaacttcaa agacaccatt gtcctcccca aggtgaggcc catgtagaga aaggatcact 300tccttgctga aagagagggt caaggggcga cccacgtggg ccctccctga aacccaggcc 360caggcctgag cctggacacc tccttccttc ctgagaccac agccagcccg gtttctctgg 420ggccaagagc aaatgctttg cttaagtgct gaaatctcag cccactgacc ccttgcagac 480aggagaggag gggaagccca gggaagctca acttcccaag tgtcctgagt ctctg 535 12 496DNA Homo sapiens 12 aatcattatg tctagcttca gtttctcctg cttcagtttaaattgggaaa gagagagaaa 60 aaatattcac tcattatctg tttcctaaaa ttgtccttaacatccttcct cttactcctt 120 tattacctgg tcgggcttcc cctcttcagg cgaaatctgtcagtctatct gcattgcctt 180 ttgatctcta cttcagttac tacmacttca aagacaccattgtcctcccc aaggtgaggs 240 ccatgtagag aaaggatcac ttccttgctg aaagagagggtcaaggggtg acccacgtgg 300 gccctccctg aaacccaggc ccaggcctga gcctggacacctccttcctt cctgagacca 360 cagccagccc ggtttctctg gggccaagag caaatgctttgcttaagtgc tgaaatctca 420 gcccactgac cccttgcmga caggagagga ggggaagcccagggaagctc aacttcccaa 480 gtgtcctgag tctctg 496 13 488 DNA Homo sapiensmisc_feature 434 n = A,T,C or G 13 tgtctagctt cagtttctcc tgcttcagtttaaattggga aagagagaga aaaaatattc 60 actcattatc tgtttcctaa aattgtccttaacatccttc ctcttactcc tttattacct 120 ggtcgggctt cccctcttca ggcgaaatctgtcagtctat ctgcattgcc ttttgatctc 180 tacttcagtt actacaactt caaagacaccattgtcctcc ccaaggtgag gcccatgtag 240 agaaaggatc acttccttgc tgaaagagagggtcaagggg tgacccacgt gggccctccc 300 tgaaacccag gcccaggcct gagcctggacacctccttcc ttcctgagac cacagccagc 360 ccggtttctc tggggccaag agcaaatgctttgcttaagt gctgaaatct cagcccactg 420 amcccttgca gacnggagag gaggggaagcccagggaagc tcaacttccc aagtgtcctg 480 agtctctg 488 14 491 DNA Homosapiens misc_feature 437 n = A,T,C or G 14 ttatgtctag cttcagtttctcctgcttca gtttaaattg ggaaagagag agaaaaaata 60 ttcactcatt atctgtttcctaaaattgtc cttaacatcc ttcctcttac tcctttatta 120 cctggtcggg cttcccctcttcaggcgaaa tctgtcagtc tatctgcatt gccttttgat 180 ctctacttca gttactacaacttcaaagac accattgtcc tccccaaggt gaggcccatg 240 tagagaaagg atcacttccttgctgaaaga gagggtcaag gggygaccca cgtgggccct 300 ccctgaaacc caggcccaggcctgagcctg gacacctcct tccttcctga gaccacagcc 360 agcccggttt ctctggggccaagagcaaat gctttgctta agtgctgaaa tctcagccca 420 ctgacccctt gcagacnggagaggagggga agcccaggga agctcaactt cccaagtgtc 480 ctgagtctct g 491 15 491DNA Homo sapiens 15 ttatgtctag cttcagtttc tcctgcttca gtttaaattgggaaagagag agaaaaaata 60 ttcactcatt atctgtttcc taaaattgtc cttaacatccttcctcttac tcctttatta 120 cctggtcggg cttcccctct tcaggcgaaa tctgtcagtctatctgcatt gccttttgat 180 ctctacttca gttactacaa cttcaaagac accattgtcctccccaaggt gaggcccatg 240 tagagaaagg atcacttcct tgctgaaaga gagggtcaaggggygaccca cgtgggccct 300 ccctgaaacc caggcccagg cctgagcctg gacacctccttccttcctga gaccacagcc 360 agcccggttt ctctggggcc aagagcaaat gctttgcttaagtgctgaaa tctcagccca 420 ctgacccctt gcagacagga gaggagggga agcccagggaagctcaactt cccaagtgtc 480 ctgagtctct g 491 16 491 DNA Homo sapiensmisc_feature 437 n = A,T,C or G 16 ttatgtctag cttcagtttc tcctgcttcagtttaaattg ggaaagagag agaaaaaata 60 ttcactcatt atctgtttcc taaaattgtccttaacatcc ttcctcttac tcctttatta 120 cctggtcggg cttcccctct tcaggcgaaatctgtcagtc tatctgcatt gccttttgat 180 ctctacttca gttactacaa cttcaaagacaccattgtcc tccccaaggt gaggcccatg 240 tagagaaagg atcacttcct tgctgaaagagagggtcaag gggygaccca cgtgggccct 300 ccctgaaacc caggcccagg cctgagcctggacacctcct tccttcctga gaccacagcc 360 agcccggttt ctctggggcc aagagcaaatgctttgctta agtgctgaaa tctcagccca 420 ctgacccctt gcagacngga gaggaggggaagcccaggga agctcaactt cccaagtgtc 480 ctgagtctct g 491 17 491 DNA Homosapiens misc_feature 159 n = A,T,C or G 17 ttatgtctag cttcagtttctcctgcttca gtttaaattg ggaaagagag agaaaaaata 60 ttcactcatt atctgtttcctaaaattgtc cttaacatcc ttcctcttac tcctttatta 120 cctggtcggg cttcccctcttcaggcgaaa tctgtcagnc tatctgcatt gccttttgat 180 ctctacttca gttactacaacttcaaagac accattgtcc tccccaaggt gaggcccatg 240 tagagaaagg atcacttccttgctgaaaga gagggtcaag gggygaccca cgtgggccct 300 ccctgaaacc caggcccaggcctgagcctg gacacctcct tccttcctga gaccacagcc 360 agcccggttt ctctggggccaagagcaaat gctttgctta agtgctgaaa tctcagccca 420 ctgacccctt gcagacaggagaggagggga agcccaggga agctcaactt cccaagtgtc 480 ctgagtctct g 491 18 487DNA Homo sapiens misc_feature 433 n = A,T,C or G 18 gtctagcttcagtttctcct gcttcagttt aaattgggaa agagagagaa aaaatattca 60 ctyattatctgtttcctaaa attgtcctta acatccttcc tcttactcct ttattacctg 120 gtcgggcttcccctcttcag gcgaaatctg tcagtctatc tgcattgcct tttgatctct 180 acttcagttactacaacttc aaagacacca ttgtcctccc caaggtgagg cccatgtaga 240 gaaaggatcacttccttgct gaaagagagg gtcaaggggy gacccacgtg ggccctccct 300 gaaacccaggcccaggcctg agcctggaca cctccttcct tcctgagacc acagccagcc 360 cggtttctctggggccaaga gcaaatgctt tgcttaagtg ctgaaatctc agcccactga 420 ccccttgcagacnggagagg aggggaagcc cagggaagct caacttccca agtgtcctga 480 gtctctg 48719 486 DNA Homo sapiens misc_feature 432 n = A,T,C or G 19 tctagcttcagtttctcctg cttcagttta aattgggaaa gagagagaaa aaatattcac 60 tcattatctgtttcctaaaa ttgtccttaa catccttcct cttactcctt tattacctgg 120 tcgggcttcccctcttcagg cgaaatctgt cagtctatct gcattgcctt ttgatctcta 180 cttcagttactacaacttca aagacaccat tgtcctcccc aaggtgaggc ccatgtagag 240 aaaggatcacttccttgctg aaagagaggg tcaaggggcg acccacgtgg gccctccctg 300 aaacccaggcccaggcctga gcctggacac ctccttcctt cctgagacca cagccagccc 360 ggtttctctggggccaagag caaatgcttt gcttaagtgc tgaaatctca gcccactgac 420 cccttgcagacnggagagga ggggaagccc agggaagctc aacttcccaa gtgtcctgag 480 tctctg 486 20484 DNA Homo sapiens misc_feature 430 n = A,T,C or G 20 tagcttcagtttctcctgct tcagtttaaa ttgggaaaga gagagaaaaa atattcactc 60 attatctgtttcctaaaatt gtccttaaca tccttcctct tactccttta ttacctggtc 120 gggcttcccctcttcaggcg aaatctgtca gtctatctgc attgcctttt gatctctact 180 tcagttactacaacttcaaa gacaccattg tcctccccaa ggtgaggccc atgtagagaa 240 aggatcacttccttgctgaa agagagggtc aaggggcgac ccacgtgggc cctccctgaa 300 acccaggcccaggcctgagc ctggacacct ccttccttcc tgagaccaca gccagcccgg 360 tttctctggggccaagagca aatgctttgc ttaagtgctg aaatctcagc ccactgaccc 420 cttgcagacnggagaggagg ggaagcccag ggaagctcaa cttcccaagt gtcctgagtc 480 tctg 484 21485 DNA Homo sapiens misc_feature 431 n = A,T,C or G 21 ctagcttcagtttctcctgc ttcagtttaa attgggaaag agagagaaaa aatattcact 60 yattatctgtttcctaaaat tgtccttaac atccttcctc ttactccttt attacctggt 120 cgggcttcccctcttcaggc gaaatctgtc agtctatctg cattgccttt tgatctctac 180 ttcagttactacaacttcaa agacaccatt gtcctcccca aggtgaggcc catgtagaga 240 aaggatcacttccttgctga aagagagggt caaggggcga cccacgtggg ccctccctga 300 aacccaggcccaggcctgag cctggacacc tccttccttc ctgagaccac agccagcccg 360 gtttctctggggccaagagc aaatgctttg cttaagtgct gaaatctcag cccactgacc 420 ccttgcagacnggagaggag gggaagccca gggaagctca acttcccaag tgtcctgagt 480 ctctg 485 22488 DNA Homo sapiens misc_feature 434 n = A,T,C or G 22 tgtctagcttcagtttctcc tgcttcagtt taaattggga aagagagaga aaaaatattc 60 acttattatctgtttcctaa aattgtcctt aacatccttc ctcttactcc tttattacct 120 ggtcgggcttcccctcttca ggcgaaatct gtcagtctat ctgcattgcc ttttgatctc 180 tacttcagttactacaactt caaagacacc attgtcctcc ccaaggtgag gcccatgtag 240 agaaaggatcacttccttgc tgaaagagag ggtcaagggg cgacccacgt gggccctccc 300 tgaaacccaggcccaggcct gagcctggac acctccttcc ttcctgagac cacagccagc 360 ccggtttctctggggccaag agcaaatgct ttgcttaagt gctgaaatct cagcccactg 420 accccttgcagacnggagag gaggggaagc ccagggaagc tcaacttccc aagtgtcctg 480 agtctctg 48823 488 DNA Homo sapiens misc_feature 434 n = A,T,C or G 23 tgtctagcttcagtttctcc tgcttcagtt taaattggga aagagagaga aaaaatattc 60 acttattatctgtttcctaa aattgtcctt aacatccttc ctcttactcc tttattacct 120 ggtcgggcttcccctcttca ggcgaaatct gtcagtctat ctgcattgcc ttttgatctc 180 tacttcagttactacaactt caaagacacc attgtcctcc ccaaggtgag gcccatgtag 240 agaaaggatcacttccttgc tgaaagagag ggtcaagggg cgacccacgt gggccctccc 300 tgaaacccaggcccaggcct gagcctggac acctccttcc ttcctgagac cacagccagc 360 ccggtttctctggggccaag agcaaatgct ttgcttaagt gctgaaatct cagcccactg 420 accccttgcagacnggagag gaggggaagc ccagggaagc tcaacttccc aagtgtcctg 480 agtctctg 48824 497 DNA Homo sapiens 24 tcactaggct tctggttgat gcctgtgaac tgaactctgacaacagactt ctgaaataga 60 cccacaagag gcagttccat ttcatttgtg ccagaatgctttaggatgta cagttatgga 120 ttgaaagttt acaggaaaaa aaattaggcc gttccttcaaagcaaatgtc ttcctggatt 180 attcaaaatg atgtatgttg aagcctttgt aaattgtcagatgctgtgca aatgttatta 240 ttttaaacat tatgatgtgt gaaaactggt taatatttataggtcacttt gttttactgt 300 cttaagttta tactcttata gacaacatgg ccgtgaactttatgctgtaa ataatcagag 360 gggaataaac tgttgagtca aaacagccat cttccttgtgaccaaacatt taaaaatatt 420 ctggctgggc acagtggctc acgcctgtaa tcccagcactttgggaggcc gaggtgggca 480 gatcacctga ggttggg 497 25 460 DNA Homo sapiens25 tgacaacaga cttctgaaat agacccacaa gaggcagttc catttcattt gtgccagaat 60gctttaggat gtacagttat ggattgaaag tttacaggaa aaaaaattag gccgttcctt 120caaagcaaat gtcttcctgg attattcaaa atgatgtatg ttgaagcctt tgtaaattgt 180cagatgctgt gcaaatgtta ttattttaaa cattatgatg tgtgaaaact ggttaatatt 240tataggtcac tttgttttac tgtcttaagt ttatactctt atagacaaca tggccgtgaa 300ctttatgctg taaataatca gaggggaata aactgttgag tcaaaacagc catcttcctt 360gtgaccaaac atttaaaaat attctggctg ggcacagtgg ctcacgcctg taatcccagc 420actttgggag gccgaggtgg gcagatcacc tgaggttggg 460 26 462 DNA Homo sapiens26 tctgacaaca gacttctgaa atagacccac aagaggcagt tccatttcat ttgtgccaga 60atgctttagg atgtacagtt atggattgaa agtttacagg aaaaaaaatt aggccgttcc 120ttcaaagcaa atgtcttcct ggattattca aaatgatgta tgttgaagcc tttgtaaatt 180gtcagatgct gtgcaaatgt tattatttta aacattatga tgtgtgaaaa ctggttaata 240tttataggtc actttgtttt actgtcttaa gtttatactc ttatagacaa catggccgtg 300aactttatgc tgtaaataat cagaggggaa taaactgttg agtcaaaaca gccatcttcc 360ttgtgaccaa acatttaaaa atattctggc tgggcacagt ggctcacgcc tgtaatccca 420gcactttggg aggccgaggt gggcagatca cctgaggttg gg 462 27 459 DNA Homosapiens 27 gacaacagac ttctgaaata gacccacaag aggcagttcc atttcatttgtgccagaatg 60 ctttaggatg tacagttatg gattgaaagt ttacaggaaa aaaaattaggccgttccttc 120 aaagcaaatg tcttcctgga ttattcaaaa tgatgtatgt tgaagcctttgtaaattgtc 180 agatgctgtg caaatgttat tattttaaac attatgatgt gtgaaaactggttaatattt 240 ataggtcact ttgttttact gtcttaagtt tatactctta tagacaacatggccgtgaac 300 tttatgctgt aaataatcag aggggaataa actgttgagt caaaacagccatcttccttg 360 tgaccaaaca tttaaaaata ttctggctgg gcacagtggc tcacgcctgtaatcccagca 420 ctttgggagg ccgaggtggg cagatcacct gaggttggg 459 28 467 DNAHomo sapiens 28 tgaactctga caacagactt ctgaaataga cccacaagag gcagttccatttcatttgtg 60 ccagaatgct ttaggatgta cagttatgga ttgaaagttt acaggaaaaaaaattaggcc 120 gttccttcaa agcaaatgtc ttcctggatt attcaaaatg atgtatgttgaagcctttgt 180 aaattgtcag atgctgtgca aatgttatta ttttaaacat tatgatgtgtgaaaactggt 240 taatatttat aggtcacttt gttttactgt cttaagttta tactcttatagacaacatgg 300 ccgtgaactt tatgctgtaa ataatcagag gggaataaac tgttgagtcaaaacagccat 360 cttccttgtg accaaacatt taaaaatatt ctggctgggc acagtggctcacgcctgtaa 420 tcccagcact ttgggaggcc gaggtgggca gatcacctga ggttggg 46729 467 DNA Homo sapiens 29 tgaactctga caacagactt ctgaaataga cccacaagaggcagttccat ttcatttgtg 60 ccagaatgct ttaggatgta cagttatgga ttgaaagtttacaggaaaaa aaattaggcc 120 gttccttcaa agcaaatgtc ttcctggatt attcaaaatgatgtatgttg aagcctttgt 180 aaattgtcag atgctgtgca aatgttatta ttttaaacattatgatgtgt gaaaactggt 240 taatatttat agrtcacttt gttttactgt cttaagtttatactcttata gacaacatgg 300 ccgtgaactt tatgctgtaa ataatcagag gggaataaactgttgagtca aaacagccat 360 cttccttgtg accaaacatt taaaaatatt ctggctgggcacagtggctc acgcctgtaa 420 tcccagcact ttgggaggcc gaggtgggca gatcacctgaggttggg 467 30 466 DNA Homo sapiens 30 gaactatgac aacagacttc tgaaatagacccacaagagg cagttccatt tcatttgtgc 60 cagaatgctt taggatgtac agttatggattgaaagttta caggaaaaaa aattaggccg 120 ttccttcaaa gcaaatgtct tcctggattattcaaaatga tgtatgttga agcctttgta 180 aattgtcaga tgctgtgcaa atgttattattttaaacatt atgatgtgtg aaaactggtt 240 aatatttata grtcactttg ttttactgtcttaagtttat actcttatag acaacatggc 300 cgtgaacttt atgctgtaaa taatcagaggggaataaact gttgagtcaa aacagccatc 360 ttccttgtga ccaaacattt aaaaatattctggctgggca cagtggctca cgcctgtaat 420 cccagcactt tgggaggccg aggtgggcagatcacctgag gttggg 466 31 466 DNA Homo sapiens 31 gaactctgac aacagacttctgaaatagac ccacaagagg cagttccatt tcatttgtgc 60 cagaatgctt taggatgtacagttatggat tgaaagttta caggaaaaaa aattaggccg 120 ttccttcaaa gcaaatgtcttcctggatta ttcaaaatga tgtatgttga agcctttgta 180 aattgtcaga tgctgtgcaaatgttattat tttaaacatt atgatgtgtg aaaactggtt 240 aatatttata grtcactttgttttactgtc ttaagtttat actcttatag acaacatggc 300 cgtgaacttt atgctgtaaataatcagagg ggaataaact gttgagtcaa aacagccatc 360 ttccttgtga ccaaacatttaaaaatattc tggctgggca cagtggctca cgcctgtaat 420 cccagcactt tgggaggccgaggtgggcag atcacctgag gttggg 466 32 460 DNA Homo sapiens 32 tgacaacagacttctgaaat agacccacaa gaggcagttc catttcattt gtgccagaat 60 gctttaggatgtacagttat ggattgaaag tttacaggaa aaaaaattag gccgttcctt 120 caaagcaaatgtcttcctgg attattcaaa atgatgtatg ttgaagcctt tgtaaattgt 180 cagatgctgtgcaaatgtta ttattttaaa cattatgatg tgtgaaaact ggttaatatt 240 tatagrtcactttgttttac tgtcttaagt ttatactctt atagacaaca tggccgtgaa 300 ctttatgctgtaaataatca gaggggaata aactgttgag tcaaaacagc catcttcctt 360 gtgaccaaacatttaaaaat attctggctg ggcacagtgg ctcacgcctg taatcccagc 420 actttgggaggccgaggtgg gcagatcacc tgaggttggg 460 33 467 DNA Homo sapiens 33tgaactctga caacagactt ctgaaataga cccacaagag gcagttccat ttcatttgtg 60ccagaatgct ttaggatgta cagttatgga ttgaaagttt acaggaaaaa aaattaggcc 120gttccttcaa agcaaatgtc ttcctggatt attcaaaatg atgtatgttg aagcctttgt 180aaattgtcag atgctgtgca aatgttatta ttttaaacat tatgatgtgt gaaaactggt 240taatatttat aggtcacttt gttttactgt cttaagttta tactcttata gacaacatgg 300ccgtgaactt tatgctgtaa ataatcagag gggaataaac tgttgagtca aaacagccat 360cttccttgtg accaaacatt taaaaatatt ctggctgggc acagtggctc acgcctgtaa 420tcccagcact ttgggaggcc gaggtgggca gatcacctga ggttggg 467 34 460 DNA Homosapiens 34 tgacaacaga cttctgaaat agacccacaa gaggcagttc catttcatttgtgccagaat 60 gctttaggat gtacagttat ggattgaaag tttacaggaa aaaaaattaggccgttcctt 120 caaagcaaat gtcttcctgg attattcaaa atgatgtatg ttgaagcctttgtaaattgt 180 cagatgctgt gcaaatgtta ttattttaaa cattatgatg tgtgaaaactggttaatatt 240 tatagatcac tttgttttac tgtcttaagt ttatactctt atagacaacatggccgtgaa 300 ctttatgctg taaataatca gaggggaata aactgttgag tcaaaacagccatcttcctt 360 gtgaccaaac atttaaaaat attctggctg ggcacagtgg ctcacgcctgtaatcccagc 420 actttgggag gccgaggtgg gcagatcacc tgaggttggg 460 35 462DNA Homo sapiens 35 tctgacaaca gacttctgaa atagacccac aagaggcagttccatttcat ttgtgccaga 60 atgctttagg atgtacagtt atggattgaa agtttacaggaaaaaaaatt aggccgttcc 120 ttcaaagcaa atgtcttcct ggattattca aaatgatgtatgttgaagcc tttgtaaatt 180 gtcagatgct gtgcaaatgt tattatttta aacattatgatgtgtgaaaa ctggttaata 240 tttatagatc actttgtttt actgtcttaa gtttatactcttatagacaa catggccgtg 300 aactttatgc tgtaaataat cagaggggaa taaactgttgagtcaaaaca gccatcttcc 360 ttgtgaccaa acatttaaaa atattctggc tgggcacagtggctcacgcc tgtaatccca 420 gcactttggg aggccgaggt gggcagatca cctgaggttg gg462 36 463 DNA Homo sapiens 36 ctctgacaac agacttctga aatagacccacaagaggcag ttccatttca tttgtgccag 60 aatgctttag gatgtacagt tatggattgaaagtttacag gaaaaaaaat taggccgttc 120 cttcaaagca aatgtcttcc tggattattcaaaatgatgt atgttgaagc ctttgtaaat 180 tgtcagatgc tgtgcaaatg ttattattttaaacattatg atgtgtgaaa actggttaat 240 atttatagrt cactttgttt tactgtcttaagtttatact cttatagaca acatggccgt 300 gaactttatg ctgtaaataa tcagaggggaataaactgtt gagtcaaaac agccatcttc 360 cttgtgacca aacatttaaa aatattctggctgggcacag tggctcacgc ctgtaatccc 420 agcactttgg gaggccgagg tgggcagatcacctgaggtt ggg 463 37 17 DNA Homo sapiens 37 ggtggctggg ctcttct 17 38 24DNA Homo sapiens 38 ctcgcttcct cagtacctat gatg 24 39 21 DNA Homo sapiens39 ctggctgagt gccagacatc t 21 40 17 DNA Homo sapiens 40 ggcgggatggagtggaa 17 41 21 DNA Homo sapiens 41 ccacctcaag ctctggtgat c 21 42 23DNA Homo sapiens 42 gttgactctt ttggcctttt cag 23 43 23 DNA Homo sapiens43 ccttaccaga cttccaggat ggt 23 44 25 DNA Homo sapiens 44 tgtccaataactgcatcacc tacct 25 45 13 DNA Homo sapiens 45 catggctgga ccc 13 46 13DNA Homo sapiens 46 catggctgga tcc 13 47 13 DNA Homo sapiens 47tgctccggcg cca 13 48 14 DNA Homo sapiens 48 ctgctctggc gcca 14 49 16 DNAHomo sapiens 49 ctctgttgcc ccagaa 16 50 15 DNA Homo sapiens 50ctctgttgcg ccaga 15 51 15 DNA Homo sapiens 51 ctttcaaggg cctgc 15 52 15DNA Homo sapiens 52 cctttcaagg ggcct 15 53 16 DNA Homo sapiens 53aagactcgag tgtcct 16 54 16 DNA Homo sapiens 54 agactcaagt gtcctc 16 55533 DNA Homo sapiens 55 ttcgtctcag tttgtttgtg agcaggctgt gagtttgggccccagaggct gggtgacatg 60 tgttggcagc ctcttcaaaa tgagccctgt cctgcctaaggctgaacttg ttttctggga 120 acaccatagg tcacctttat tctggcagag gagggagcatcagtgccctc caggatagac 180 ttttcccaag cctacttttg ccattgactt cttcccaagattcaatccca ggatgtacaa 240 ggacagcccc tcctccatag tatgggactg gcctctgctgatcctcccag gcttccgtgt 300 gggtcagtgg ggcccatgga tgtgcttgtt aactgagtgccttttggtgg agaggcccgg 360 cctctcacaa aagacccctt accactgctc tgatgaagaggagtacacag aacacataat 420 tcaggaagca gctttcccca tgtctcgact catccatccaggccattccc cgtctctggt 480 tcctcccctc ctcctggact cctgcacacg ctccttcctctgaggctgaa att 533 56 497 DNA Homo sapiens 56 gggccccaga ggctgggtgacatgtgttgg cagcctcttc aaaatgagcc ctgtcctgcc 60 taaggctgaa cttgttttctgggaacacca taggtcacct ttattctggc agaggaggga 120 gcatcagtgc cctccaggatagacttttcc caagcctact tttgccattg acttcttccc 180 aagattcaat cccaggatgtacaaggacag cccctcctcc atagtatggg actggcctct 240 gctgatcctc ccaggcttccgtgtgggtca gtggggccca tggatgtgct tgttaactga 300 gtgccttttg gtggagaggcccggcctctc acaaaagacc ccttaccact gctctgatga 360 agaggagtac acagaacacataattcagga agcagctttc cccatgtctc gactcatcca 420 tccaggccat tccccgtctctggttcctcc cctcctcctg gactcctgca cacgctcctt 480 cctctgaggc tgaaatt 49757 497 DNA Homo sapiens 57 gggccccaga ggctgggtga catgtgttgg aagcctcttcaaaatgagcc ctgtcctgcc 60 taaggctgaa cttgttttct gggaacacca taggtcacctttattctggc agaggaggga 120 gcatcagtgc cctccaggat agacttttcc caagcctacttttgccattg acttcttccc 180 aagattcaat cccaggatgt acaaggacag cccctcctccatagtatggg actggcctct 240 gctgatcctc ccaggcttcc gtgtgggtca gtggggcccatggatgtgct tgttaactga 300 gtgccttttg gtggagaggc ccggcctctc acaaaagaccccttaccact gctctgatga 360 agaggagtac acagaacaca taattcagga agcagctttccccatgtctc gactcatcca 420 tccaggccat tccccgtctc tggttcctcc cctcctcctggactcctgca cacgctcctt 480 cctctgaggc tgaaatt 497 58 497 DNA Homo sapiens58 gggccccaga ggctgggtga catgtgttgg cagcctcttc aaaatgagcc ctgtcctgcc 60taaggctgaa cttgttttct gggaacacca taggtcacct ttattctggc agaggaggga 120gcatcagtgc cctccaggat agacttttcc caagcctact tttgccattg acttcttccc 180aagattcaat cccaggatgt acaaggacag cccctcctcc atagtatggg actggcctct 240gctgatcctc ccaggcttcc gtgtgggtca gtggggccca tggatgtgct tgttaactga 300gtgccttttg gtggagaggc ccggcctctc acaaaagacc ccttaccact gctctgatga 360agaggagtac acagaacaca taattcagga agcagctttc cccatgtctc gactcatcca 420tccaggccat tccccgtctc tggttcctcc cctcctcctg gactcctgca cacgctcctt 480cctctgaggg tgaaatt 497 59 483 DNA Homo sapiens 59 gggtgacatg tgttggcagcctcttcaaaa tgagccctgt cctgcctaag gctgaacttg 60 ttttctggga acaccataggtcacctttat tctggcagag gagggagcat cagtgccctc 120 caggatagac ttttcccaagcctacttttg ccattgactt cttcccaaga ttcaatccca 180 ggatgtacaa ggacagcccctcctccatag tatgggactg gcctctgctg atcctcccag 240 gcttccgtgt gggtcagtggggcccatgga tgtgcttgtt aactgagtgc cttttggtgg 300 agaggcccgg cctctcacaaaagacccctt accactgctc tgatgaagag gagtacacag 360 aacacmtaat tcaggaagcagctttcccca tgtctcgact catccatcca ggccattccc 420 cgtctctggt tcctcccctcctcctggact cctgcacacg ctccttcctc tgaggctgaa 480 att 483 60 500 DNA Homosapiens 60 tttgggcccc agaggctggg tgacatgtgt tggcagcctc ttcaaaatgagccctgtcct 60 gcctaaggct gaacttgttt tctgggaaca ccataggtca cctttattctggcagaggag 120 ggagcatcag tgccctccag gatagacttt tcccaagcct acttttgccattgacttctt 180 cccaagattc aatcccagga tgtacaagga cagcccctcc tccatagtatgggactggcc 240 tctgctgatc ctcccaggct tccgtgtggg tcagtggggc ccatggatgtgcttgttaac 300 tgagtgcctt ttggtggaga ggcccggcct ctcacaaaag accccttmccactgctctga 360 tgaagaggag tacacagaac acataattca ggaagcagct ttccccatgtctcgactcat 420 ccatccaggc cattccccgt ctctggttcc tcccctcctc ctggactcctgcacacgctc 480 cttcctctga ggctgaaatt 500 61 499 DNA Homo sapiens 61ttgggcccca gaggctgggt gacatgtgtt ggcagcctct tcaaaatgag ccctgtcctg 60cctaaggctg aacttgtttt ctgggaacac cataggtcac ctttattctg gcagaggagg 120gagcatcagt gccctccagg atagactttt cccaagccta cttttgccat tgacttcttc 180ccaagattca atcccaggat gtacaaggac agcccctcct ccatagtatg ggactggcct 240ctgctgatcc tcccaggctt ccgtgtgggt cagtggggcc catggatgtg cttgttaact 300gagtgccttt tggtggagag gcccggcctc tcacaaaaga ccccttmcca ctgctctgat 360gaagaggagt acacagaaca cataattcag gaagcagctt tccccatgtc tcgactcatc 420catccaggcc attccccgtc tctggttcct cccctcctcc tggactcctg cacacgctcc 480ttcctctgag gctgaaatt 499 62 498 DNA Homo sapiens 62 tgggccccagaggctgggtg acatgtgttg gcagcctctt caaaatgagc cctgtcctgc 60 ctaaggctgaacttgttttc tgggaacacc ataggtcacc tttattctgg cagaggaggg 120 agcatcagtgccctccagga tagacttttc ccaagcctac ttttgccatt gacttcttcc 180 caagattcaatcccaggatg tacaaggaca gcccctcctc catagtatgg gactgggctc 240 tgctgatcctcccaggcttc cgtgtgggtc agtggggccc atggatgtgc ttgttaactg 300 agtgccttttggtggagagg cccggcctct cacaaaagac cccttmccac tgctctgatg 360 aagaggagtacacagaacac ataattcagg aagcagcttt ccccatgtct cgactcatcc 420 atccaggccattccccgtct ctggttcctc ccctcctcct ggactcctgc acacgctcct 480 tcctctgaggctgaaatt 498 63 498 DNA Homo sapiens 63 tgggccccag aggctgggtg acatgtgttggcagcctctt caaaatgagc cctgtcctgc 60 ctaaggctga acttgttttc tgggaacaccataggtcacc tttattctgg cagaggaggg 120 agcatcagtg ccctccagga tagacttttcccaagcctac ttttgccatt gacttcttcc 180 caagattcaa tcccaggatg tacaaggacagcccctcctc catagtatgg gactggcctc 240 tgctgatcct cccaggcttc cgtgtgggtcagtggggccc atggatgtgc ttgttaactg 300 agtgcctttt ggtggagagg cccggcctctcacaaaagac cccttmccac tgctctgatg 360 aagaggagta cacagaacac ataattcaggaagcagcttt ccccatgtct cgactcatcc 420 atccaggcca ttccccgtct ctggttcctcccctcctcct ggactcctgc acacgctcct 480 tcctctgagg ctgaaatt 498 64 498 DNAHomo sapiens 64 tgggccccag aggctgggtg acatgtgttg gcagcctctt caaaatgagccctgtcctgc 60 ctaaggctga acttgttttc tgggaacacc ataggtcacc tttattctggcagaggaggg 120 agcatcagtg ccctccagga tagacttttc ccaagcctac ttttgccattgacttcttcc 180 caagattcaa tcccaggatg tacaaggaca gcccctcctc catagtatgggactggcctc 240 tgctgatcct cccaggcttc cgtgtgggtc agtggggccc atggatgtgcttgttaactg 300 agtgcctttt ggtggagagg cccggcctct cacaaaagac cccttaccactgctctgatg 360 aagaggagta cacagaacac ataattcagg aagcagcttt ccccatgtctcgactcatcc 420 atccaggcca ttccccgtct ctggttcctc ccctcctcct ggactcctgcacacgctcct 480 tcctctgagg ctgaaatt 498 65 503 DNA Homo sapiens 65gagtttgggc cccagaggct gggtgacatg tgttggcagc ctcttcaaaa tgagccctgt 60cctgcctaag gctgaacttg ttttctggga acaccatagg tcacctttat tctggcagag 120gagggagcat cagtgccctc caggatagac ttttcccaag cctacttttg ccattgactt 180cttcccaaga ttcaatccca ggatgtacaa ggacagcccc tcctccatag tatgggactg 240gcctctgctg atcctcccag gcttccgtgt gggtcagtgg ggcccatgga tgtgcttgtt 300aactgagtgc cttttggtgg agaggcccgg cctctcacaa aagacccctt cccactgctc 360tgatgaagag gagtacacag aacacataat tcaggaagca gctttcccca tgtctcgact 420catccatcca ggccattccc cgtctctggt tcctcccctc ctcctggact cctgcacacg 480ctccttcctc tgaggctgaa att 503 66 453 DNA Homo sapiens 66 tgagccctgtcctgcctaag gctgaacttg ttttctggga acaccatagg tcacctttat 60 tctggcagaggagggagcat cagtgccctc caggatagac ttttcccaag cctacttttg 120 ccattgacttcttcccaaga ttcaatccca ggatgtacaa ggacagcccc tcctccatag 180 tatgggactggcctctgctg atcctcccag gcttccgtgt gggtcagtgg ggcccatgga 240 tgtgcttgttaactgagtgc cttttggtgg agaggcccsg cctctcacaa aagacccctt 300 cccactgctctgatgaagag gagtacacag aacacataat tcaggaagca gctttcccca 360 tgtctcgactcatccatcca ggccattccc cgtctctggt tcctcccctc ctcctggact 420 cctgcacacgctccttcctc tgaggctgaa att 453 67 500 DNA Homo sapiens 67 tttgggccccagaggctggg tgacatgtgt tggcagcctc ttcaaaatga gccctgtcct 60 gcctaaggctgaacttgttt tctgggaaca ccataggtca cctttattct ggcagaggag 120 ggagcatcagtgccctccag gatagacttt tcccaagcct acttttgcca ttgacttctt 180 cccaagattcaatcccagga tgtacaagga cagcccctcc tccatagtat gggactggcc 240 tctgctgatcctcccaggct tccgtgtggg tcagtggggc ccatggatgt gcttgttaac 300 tgagtgccttttggtggaga ggcccggcct ctcacaaaag accccttmcc actgctctga 360 tgaagaggagtacacagaac acataattca ggaagcagct ttccccatgt ctcgactcat 420 ccatccaggccattccccgt ctctggttcc tcccctcctc ctggactcct gcacacgctc 480 cttcctctgagggtgaaatt 500

We claim:
 1. A method of diagnosing or predicting susceptibility to aclinical subtype of Crohn's disease characterized by fibrostenosingdisease, comprising determining the presence or absence in an individualof a fibrostenosis-predisposing allele linked to a NOD2/CARD15 locus,wherein the presence of said fibrostenosis-predisposing allele isdiagnostic of or predictive of susceptibility to the clinical subtype ofCrohn's disease characterized by fibrostenosing disease.
 2. The methodof claim 1, wherein said clinical subtype of Crohn's disease ischaracterized by fibrostenosing disease independent of small bowelinvolvement.
 3. The method of claim 1, wherein saidfibrostenosis-predisposing allele is located within said NOD2/CARD15locus.
 4. The method of claim 3, wherein NF-kappa B activation by aNOD2/CARD15 polypeptide encoded by said fibrostenosis-predisposingallele is reduced as compared to NF-kappa B activation by a wild-typeNOD2/CARD15 polypeptide.
 5. The method of claim 3, wherein saidfibrostenosis-predisposing allele is located in a coding region of saidNOD2/CARD15 locus.
 6. The method of claim 5, wherein saidfibrostenosis-predisposing allele is located in a region encodingresidues 744 to 1020 of NOD2/CARD15.
 7. The method of claim 5, whereinsaid fibrostenosis-predisposing allele is a “2” allele at a SNP selectedfrom SNP 8, SNP 12, and SNP
 13. 8. The method of claim 7, wherein saidfibrostenosis-predisposing allele is a “2” allele at SNP
 13. 9. Themethod of claim 3, wherein said fibrostenosis-predisposing allele islocated in a non-coding region of said NOD2/CARD15 locus.
 10. The methodof claim 9, wherein said fibrostenosis-predisposing allele is selectedfrom a JW1, JW15, and JW16 variant allele.
 11. The method of claim 9,wherein said fibrostenosis-predisposing allele is located in a promoterregion of said NOD2/CARD15 locus.
 12. The method of claim 11, whereinsaid fibrostenosis-predisposing allele is an allele selected from a JW17and JW18 variant allele.
 13. The method of claim 1, comprisingdetermining the presence or absence in said individual of at least twofibrostenosis-predisposing alleles linked to a NOD2/CARD15 locus,wherein the presence of one or more of said fibrostenosis-predisposingalleles is diagnostic of or predictive of susceptibility to the clinicalsubtype of Crohn's disease characterized by fibrostenosing disease. 14.The method of claim 13, wherein said at least twofibrostenosis-predisposing alleles are “2” alleles at a SNP selectedfrom SNP 8, SNP 12, and SNP
 13. 15. The method of claim 14, comprisingdetermining the presence or absence in said individual of (i) a “2”allele at SNP 8, (ii) a “2” allele at SNP 12, and (iii) a “2” allele atSNP 13, wherein the presence of one or more of said “2” alleles at SNP8, SNP 12, and SNP 13 is diagnostic of or predictive of susceptibilityto the clinical subtype of Crohn's disease characterized byfibrostenosing disease.
 16. The method of claim 1, wherein saidfibrostenosis-predisposing allele is associated with said clinicalsubtype of Crohn's disease characterized by fibrostenosing disease withan odds ratio of at least 2 and a lower 95% confidence limit greaterthan
 1. 17. The method of claim 1, further comprising generating areport indicating the presence or absence in said individual of saidfibrostenosis-predisposing allele.
 18. The method of claim 1, furthercomprising generating a report indicating the presence or absence insaid individual of said clinical subtype of Crohn's diseasecharacterized by fibrostenosing disease.
 19. The method of claim 1,wherein determining the presence or absence of saidfibrostenosis-predisposing allele comprises enzymatic amplification ofnucleic acid from said individual.
 20. The method of claim 19, whereinsaid amplification is polymerase chain reaction amplification.
 21. Themethod of claim 20, wherein said polymerase chain reaction amplificationis performed using one or more fluorescently labeled probes.
 22. Themethod of claim 20, wherein said polymerase chain reaction amplificationis performed using one or more probes comprising a DNA minor grovebinder.
 23. A method of optimizing therapy in an individual, comprising(a) determining the presence or absence in said individual of afibrostenosis-predisposing allele linked to a NOD2/CARD15 locus, (b)diagnosing individuals in which said fibrostenosis-predisposing alleleis present as having a fibrostenosing subtype of Crohn's disease, and(c) treating said individual having a fibrostenosing subtype of Crohn'sdisease based on said diagnosis.